Abstract:
Tube and float systems for analyzing target materials of a suspension include in which at least a portion of the outer surface of the float is reflective are described. The target material particles can be conjugated with fluorophores. In order to identify the target material, the material between the float and tube is illuminated with one or more channels of excitation radiation, which causes the fluorophores to become excited and emit radiation at longer wavelengths. The reflective surface of the float reflects the excitation radiation and the emitted radiation.

Description:
CROSS-REFERENCE TO A RELATED APPLICATION 
     This application claims the benefit of Provisional Application No. 61/577,866, filed Dec. 20, 2011. 
    
    
     TECHNICAL FIELD 
     This disclosure relates generally to density-based fluid separation and, in particular, to tube and float systems for the separation and axial expansion of constituent suspension components layered by centrifugation. 
     BACKGROUND 
     Suspensions often include materials of interest that are difficult to detect, extract and isolate for analysis. For instance, whole blood is a suspension of materials in a fluid. The materials include billions of red and white blood cells and platelets in a proteinaceous fluid called plasma. Whole blood is routinely examined for the presence of abnormal organisms or cells, such as 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 labeling the film in a way that enables certain components to be examined by bright field microscopy. 
     On the other hand, materials of interest composed of particles that occur in very low numbers are especially difficult if not impossible to detect and analyze using many existing techniques. Consider, for instance, circulating tumor cells (“CTCs”), which are cancer cells that have detached from a tumor, circulate in the bloodstream, and may be regarded as seeds for subsequent growth of additional tumors (i.e., metastasis) in different tissues. The ability to accurately detect and analyze CTCs is of particular interest to oncologists and cancer researchers, but CTCs occur in very low numbers in peripheral whole blood samples. For instance, a 7.5 ml sample of peripheral whole blood that contains as few as 5 CTCs is considered clinically relevant in the diagnosis and treatment of a cancer patient. However, detecting even 1 CTC in a 7.5 ml blood sample is equivalent to detecting 1 CTC in a background of about 40 billion red and white blood cells. Using existing techniques to find, isolate and extract as few as 5 CTCs of a whole blood sample is extremely time consuming, costly and may be impossible to accomplish. As a result, practitioners, researchers, and those working with suspensions continue to seek systems and methods to more efficiently and accurately detect, isolate and extract target materials of a suspension. 
     SUMMARY 
     Systems for analyzing target materials of a suspension include a tube and a float in which at least a portion of the outer surface of the float is reflective. The target material particles are conjugated with fluorophores, and the tube, float and suspension are centrifuged so that at least a portion of the target material is located between the inner wall of the tube and the reflective surface of the float. In order to identify the target material, the material between the float and tube is illuminated with one or more channels of excitation radiation, which causes the fluorophores to become excited and emit radiation at longer wavelengths. The reflective surface of the float reflects the excitation radiation and the emitted radiation. As a result, the reflected excitation radiation may excite fluorophores that are not able to be directly illuminated by the excitation radiation and the reflected emitted radiation increases the total intensity of the emitted radiation. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A-1B  show isometric views of two example tube and float systems. 
         FIGS. 2A-2D  shows four examples of floats with different types of structural elements and end caps. 
         FIGS. 3A-3C  show three example reflective floats. 
         FIGS. 4A-4B  show an example tube and reflective float system used with a suspension containing a target material. 
         FIG. 5  shows a cross-sectional view of the tube and reflective float system along a line I-I shown in  FIG. 4B . 
         FIG. 6A  shows a black and white image of a first fluorescently labeled cell using a tube and non-reflective float system. 
         FIG. 6B  shows a black and white image of a second fluorescently labeled cell using a tube and reflective float system. 
         FIG. 7  shows four gray scale intensity versus distance plots, each plot associated with a cell labeled with a different fluorophore. 
     
    
    
     DETAILED DESCRIPTION 
     The detailed description is organized into four subsections: A general description of tube and float systems is provided in a first subsection. Examples of reflective floats are provided in a second subsection. Using tube and reflective float systems to analyze target materials of a suspension is provided in a third subsection. And experimental results that contrast performance of a white float with a black float are presented in a fourth subsection. 
     In the following description, the term “light” is used to describe various uses and aspects of tube and reflective float systems. The term light is not intended to be limited to describing electromagnetic radiation in the visible portion of the electromagnetic spectrum, but is also intended to describe radiation in the ultraviolet and infrared portions of the electromagnetic spectrum. 
     General Description of Tube and Float Systems 
       FIG. 1A  shows an isometric view of an example tube and float system  100 . The system  100  includes a tube  102  and a float  104  suspended within a suspension  106 . In the example of  FIG. 1A , the tube  102  has a circular cross-section, a first closed end  108 , and a second open end  110 . The open end  110  is sized to receive a stopper or cap  112 . A tube may also have two open ends that are sized to receive stoppers or caps, such as the tube  122  of an example tube and float system  120  shown  FIG. 1B . The system  120  is similar to the system  100  except the tube  102  of the system  100  is replaced by a tube  122  that includes two open ends  124  and  126  configured to receive the cap  112  and a cap  128 , respectively. The tubes  102  and  122  have a generally cylindrical geometry, but may also have a tapered geometry that widens toward the open ends  110  and  124 , respectively. Although the tubes  102  and  122  have a circular cross-section, in other embodiments, the tubes  102  and  122  can have elliptical, square, triangular, rectangular, octagonal, or any other suitable cross-sectional shape that substantially extends the length of the tube. The tubes  102  and  122  can be composed of a transparent or semitransparent flexible material, such as flexible plastic or another suitable material. 
       FIGS. 2A-2D  shows four examples of floats  104  and  201 - 203  with different types of structural elements and end caps. In  FIG. 2A , the float  104 , shown in  FIG. 1 , includes a main body  204 , a cone-shaped end cap  206 , a dome-shaped end cap  208 , and structural elements in the form of splines  210  that are radially spaced and axially oriented. The splines  210  provide a sealing engagement with the inner wall of the tube  102 . In other embodiments, the number of splines, spline spacing, and spline thickness can be independently varied. The splines  210  can also be broken or segmented. The main body  204  is sized to have an outer diameter that is less than the inner diameter of the tube  102 , thereby defining fluid retention channels between the outer surface of the body  204  and the inner wall of the tube  102 . The outer surfaces of the body  204  between the splines  210  can be flat, curved or have another suitable geometry. In the example of  FIG. 2A , the splines  210  and the body  204  form a single structure. Embodiments include other types of geometric shapes for float end caps. In  FIG. 2B , an example float  201  has two cone-shaped end caps  212  and  214 . The main body  216  of the float  201  includes the same structural elements (i.e., splines) as the float  104 . A float can also include two dome-shaped end caps. Float end caps can be configured with other geometric shapes and are not intended to be limited to the shapes described herein. In other embodiments, the main body of a float can include a variety of different structural elements for separating target materials, supporting the tube wall, or directing the suspension fluid around the float during centrifugation.  FIGS. 2C and 2D  show examples of two different types of main body structural elements. Embodiments are not intended to be limited to these two examples. In  FIG. 2C , the main body  218  of the float  202  is similar to the float  104  except the main body  218  includes a number of protrusions  220  that provide support for the deformable tube. In other embodiments, the number and pattern of protrusions can be varied. In  FIG. 2D , the main body  222  of the float  203  includes a single continuous helical structure or ridge  224  that spirals around the main body  222  creating a helical channel  226 . In other embodiments, the helical ridge  224  can be rounded or broken or segmented to allow fluid to flow between adjacent turns of the helical ridge  224 . In other embodiments, the helical ridge spacing and rib thickness can be independently varied. 
     The floats can be composed of a rigid organic or inorganic materials, and rigid 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 and others. 
     Examples of Reflective Floats 
     The floats described above with reference to  FIGS. 1 and 2  are shaded to represent dark colored floats. When a dark colored float is used to trap a target material between the outer surface of the float and the inner wall of a tube and the target material is imaged by illuminating the material the float and the tube. But a dark colored float may absorb an appreciable amount of the light, which may cause the float to heat up and expand within the tube, which, in turn, may cause target material particles to change position. This shift in particle position makes it difficult and time consuming to relocate target particles because the particles have randomly moved to different locations. In order to reduce the amount by which target particles shift when the target material is illuminated, a reflective float with a reflective outer surface can be used.  FIGS. 3A-3C  show three different example reflective floats  301 - 303 . The reflective floats  301 - 303  are similar to the float  104  except the floats  301 - 303  include one or more reflective surfaces represented by unshaded regions. In  FIG. 3A , the entire outer surface of the float  301  is reflective. In  FIG. 313 , the outer surface of main body portion  304 , including the structural elements or splines  306 , is a reflective surface. In  FIG. 3C , outer surfaces of main body portion  308 , excluding structural elements or splines  310 , are reflective surfaces. 
     A reflective float can be created by adding a highly reflective or white pigment to the material composition of the float. A reflective float can be created by combining the rigid organic and inorganic materials listed above with a white pigment during fabrication of the float. As a result, the entire float is reflective, as shown in the example of  FIG. 3A . The float can have a glossy or matte finish. Examples of white plastics that can be used for a reflective float include, but are not limited to, white Delrin®, moisture resistance polyester, wear-resistant slippery cast nylon 6, impact-resistant slippery UHMW polyethylene, opaque white polypropylene, rigid HDPE polyethylene, UV resistant VHMW polyethylene, acrylic PVC, flame-retardant polypropylene, moisture-resistant LDPE polyethylene, lightweight rigid PVC foam, structural fiberglass, and white polystyrene. A reflective surface of a float can be a reflective coating applied to the outer surface of the float. For example, the coating can be a reflective paint, such as white paint, paint with reflective particles or ceramic beads or a reflective polymer. The paint can have a glossy or matte finish. A reflective surface of a float can be a reflective plating applied to the outer surface of the float. For example, the plating can be a shiny reflective metal, ceramic, or a mirror. Suitable reflective metals include, but are not limited to, gold, silver, aluminum, tin, copper, bronze, chromium, cobalt, nickel, palladium, platinum, manganese, zinc, titanium, niobium, molybdenum, tungsten, or a suitable metalloid. The reflective outer surface can also be created by reflective objects or particles attached to the outer surfaces of the float using an adhesive or embedded in the outer surface of the float. 
     Using Tube and Reflective Float Systems to Analyze Target Materials of a Suspension 
       FIGS. 4A-4B  show an example tube and reflective float system  400  used with a suspension containing a target material. The system  400  is similar to the system  100  describe above with reference to  FIG. 1  except the float  104  has been replaced by the reflective float  302 . In the example of  FIG. 4A , the reflective float  302  is deposited in the tube  102  along with a suspension  404  that contains a target material. The float  302  has a density that approximately matches the density of the target material. Prior to introducing the float  302  to the tube  102 , the suspension is mixed with a solution that contains fluorescent probes. The fluorescent probes can be fluorescent chemical compounds called “fluorophores” attached to ligands that selectively bind to the target material particles. In other words, the target material particles are fluorescently labeled. When the tube  102 , float  302  and suspension  404  are centrifuged together for a period of time, the suspension materials separate into layers along the axial length of the tube according the density associated with each layer with higher density materials settling beneath lower density materials. In the example of  FIG. 4B , centrifugation is used to separate the suspension  404  into three layers  406 - 408 . Any unattached fluorescent probes are present in the top layer  408  above the float  302 . Because the float  302  has a density that approximately matches the density of the target material, the float  302  is positioned at approximately the same level as the layer  407  and expands the axial length of the layer  407  between the main body of the float  302  and the inner wall of the tube  102 . The layer  407  contains the target material with fluorescently labeled target material particles located within the channels between the main body of the float  302  and the inner wall of the tube  102 . 
     The suspension  404  can be a biological suspension, such as whole blood, stool, semen, cerebrospinal fluid, nipple aspirate fluid, saliva, amniotic fluid, vaginal secretions, mucus membrane secretions, aqueous humor, vitreous humor, vomit, and any other physiological fluid or semi-solid. The target material particles can be cells, such as ova or circulating tumor cells, parasites, microorganisms, or inflammatory cells. The target particles may have a number of different types of receptor molecules located on the surface. Each type of receptor is a molecule capable of attaching a particular ligand. Examples of ligands include peptides, neurotransmitters, hormones, pharmaceutical drugs, toxins, and other types of molecules. As a result, ligands can be used to classify the target particles and determine the specific type of target particles present in the suspension by conjugating ligands that attach to particular receptors with a particular fluorophore. For example, each type of fluorophore emits light in a narrow wavelength range of the electromagnetic spectrum called a “channel” when an appropriate stimulus, such as light with a shorter wavelength, is applied. A first type of fluorophore that emits light in the green channel can be attached to a first ligand that binds specifically to a first type of receptor, while a second type of fluorophore that emits light in the red channel can be attached to a second ligand that binds specifically to a second type of receptor. The channel color observed as a result of stimulating the target material identifies the type of receptor, and because receptors can be unique to particular target particles, the channel color can also be used to identify the target particle. Examples of suitable fluorophores include, but are not limited to, fluorescein, FITC (“fluorescein isothiocyanate”), phycoerythrin, Cy5PE, Cy7PE, Texas Red, allophycocyanin, Cy5, Cy7APC, cascade blue, biotin, DAPI (“4′,6-diamidino-2-phenylindole”) and TRITC (“tetramethylrhodamine isothiocyanate”). 
     In practice, a suspension including a target material is added to a tube along with a reflective float and the tube, float and suspension are centrifuged to trap target material particles between the float and the tube, as described above. The target particles can be fixed, permeabilized, and labeled. The target material is imaged by illuminating the tube with different wavelengths of excitation light that excite the different fluorophores. The light emitted from the excited fluorophores is captured in images by a scanner camera, allowing the target particles to be enumerated identified based on the channel of the light emitted. 
     The reflective surface of a reflective float can increase the intensity of light emitted from the fluorophores.  FIG. 5  shows a cross-sectional view of the system  400  along a line I-I shown in  FIG. 4B . In the example of  FIG. 5 , the cross-sectional view reveals that the main body  304  of the reflective float  302  is coated with a reflective layer  502 . A multichannel light source  504  illuminates the target material in a channel  506  with excitation light to excite fluorophores attached to target material particles.  FIG. 5  includes a magnified view  508  of a portion of the channel  506 . Circle  510  represents a target material particle and smaller shaded circles, such as circle  512 , represent six fluorophores attached to the particle  510  via ligands. Solid-line directional arrows  514 - 518  represent rays of excitation light associated with a channel output from the source  504 . As shown in  FIG. 5 , rays  515 - 517  pass through the tube  102  to illuminate the fluorophores facing the tube  102 . Dashed-line directional arrows  520 - 522  represent rays of light emitted from the fluorophores that face the tube  102 . Rays  514  and  518  represent excitation light that is reflected off of the reflective layer  502  to illuminate fluorophores that face the float  302 . Dashed-line directional arrows  523  and  524  represent rays of excitation light emitted from the fluorophores that face the float  302 . 
     Note that without the reflective layer  502 , much of the light represented by the rays  514  and  518  is absorbed by the float  302  and is not available to excite the fluorophores that face the float  302 . In the example of  FIG. 5 , the light emitted from the fluorophores that face the float  302  is also reflected from the reflective layer  502  and adds to the intensity of the light emitted from the fluorophores that face the tube  102 . As a result, images of the particle  510  appear brighter than the particle  510  would otherwise appear with a dark colored or non-reflective float. Note also that because the excitation light and the emitted light are not absorbed by the float  302 , the float  302  does not heat up and expand. As a result, the particle  510  is less likely to shift, making it easier to identify the location of the particle  510  and the same particle  510  can be relocated when the target material is illuminated a second time. 
     The reflective surface of a reflective float can also be functionalized to attract or attach target particles to the float. The reflective surface may be functionalized using a self-assembled monolayer comprising a head, a tail, and a functional group. The head reacts with and attaches to the reflective surface, and may be any chemical having a high affinity for the reflective surface. For example, sulfur has a high affinity for metals. The tail can be a carbon backbone that connects the head to the functional group and may be any suitable length and may or may not be branched. The functional group is selected based on the appropriate functionality or reaction desired. Examples of self-assembled monolayers include alkanethiols for metals and silanes for nonmetallic oxides. After the reflective surface has been functionalized, materials may be added the suspension to provide better capture of the target particles. The materials include Mytilus edulis foot protein (“Mefp”); biopolymers; polyphenolic proteins (including those polyphenolic proteins containing L-DOPA); chemo-attractant molecules, such as epidermal growth factor (“EGF”) or vascular endothelial growth factor (“VEGF”); an extracellular matrix protein (“ECM”); maleic anhydride; maleimide activated sulfa-hydryl groups, poly-L-lysine; poly-D-lysine; streptavidin; neutravidin; protein A; protein G; protein AIG, protein L; biotin; glutathione; antibodies; recombinant antibodies; aptamers; RGD-peptides; fibronectin; collagen; elastin; fibrillin; laminin; or proteoglycans. 
     Experimental Results 
     Experimental results obtained from comparing a reflective float composed of white Deirin® float and a black float composed of black Deirin® are now described. The floats were placed in separate tubes, each with a whole blood sample containing circulating tumor cells (“CTCs”). The tube and float systems were centrifuged and imaged as described above.  FIG. 6A  shows a black and white image of a first CTC labeled with TRITC using a tube and float system in which the float is composed of black Deirin®.  FIG. 6B  shows a black and white image of a second CTC also labeled with TRITC but the float of the tube and float system is composed of white Delrin®.  FIGS. 6A and 6B  both include gray scale intensity versus distance plots  601  and  602 . The plot  601  represents the recorded intensity versus distance for the first cell along a white line, shown in  FIG. 6A , that bisect the first cell. The plot  602  represents the recorded intensity versus distance for the second cell along a white line, shown in  FIG. 6B , that bisect the second cell. The image of the first CTC and the intensity plot  601  where obtained for an exposure time of approximately 0.05 seconds, and the image of the second CTC and intensity plot  602  where obtained for an exposure time of approximately 0.2 seconds. In  FIG. 6A , regions  604  and  605  of the plot  601  correspond to background light and hump  606  represents the intensity across the first CTC along the corresponding white line. The jagged appearance of the intensity in plot  601  represents the graininess of the image shown in  FIG. 6A . Plot  601  reveals that the intensity of the first CTC above the background ranges from about 50 to about 450, with the greatest intensity appearing near the middle of the first CTC. In  FIG. 6B , regions  608  and  609  of the plot  602  correspond to background light, while hump  610  represents the intensity across the second CTC along the corresponding white line. Plot  602  reveals that the intensity of the second CTC above the background ranges from about 1000 to about 4500, with the greatest intensities occurring around the perimeter CTC image. In the image of  FIG. 6B , the second CTC has a brighter perimeter than the first CTC shown in  FIG. 6A  because more TRITC labels located around the perimeter of the second CTC are excited by the excitation light reflected off of the white float. In other words, the signal-to-noise ratio is greater for the white float than for the black float. 
       FIG. 7  shows four different gray scale intensity plots  701 - 704  associated with four cells, each plot corresponds to a different fluorophore. Plots  701 - 704  were obtained for CTCs of a whole blood sample labeled with DAPI, FITC, TRITC, and Cy5, respectively. Dashed-line curves, such as curve  706 , represent the gray scale intensity across the cells obtained from tube and float systems in which the floats were composed of white Delrin®. Solid-line curves, such as curve  708 , represent the gray scale intensity across cells obtained from tube and float systems in which the floats were composed of black Delrin®. The peaks, such as peak  710 , represent the intensity of the light emitted from the fluorescent labels of the cells and flat regions surrounding the peaks represent the intensity of the background light. Plots  701 - 704  all reveal that the intensity difference between the cells and the associated backgrounds were greater for the white float than for the black float. In other words, for the four types of fluorophores analyzed, the white float produces a larger signal-to-noise ratio than the black float. 
     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: