Patent Publication Number: US-2011067488-A1

Title: Systems and methods for reducing expansion of fluid contraining tubes during centrifugation

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of Provisional Application No. 61/241,133, filed Sep. 10, 2009. 
    
    
     TECHNICAL FIELD 
     The present invention relates to centrifuging tubes. 
     BACKGROUND 
     Tubes containing a suspension are typically centrifuged at high speeds to separate the particles of the suspension into layers according to their respectively specific gravities. High speed centrifugation of a tube containing a suspension creates an outward, radially directed, hydrostatic force on the tube. At the maximum distance from the rotational center of the centrifuge the hydrostatic force is greatest. As the distance from the centrifuge axis decreases, the hydrostatic force decreases linearly but remains substantial throughout most of the tube. The hydrostatic force is often sufficient to expand the diameter of the tube during centrifugation. As the centrifuge slows to a stop, the tube often returns to its pre-expansion size, provided the tube&#39;s elastic limit is not exceeded. 
     While this expansion is normally of little concern in many routine industrial and laboratory procedures, it can be of considerable significance when attempting to identify and isolate rare particles in a suspension. For example, as the tube returns to its pre-expansion size as centrifugation stops, abundant particles in a layer adjacent to a layer containing the rare particles may shift into the rare particle layer preventing identification and isolation of the rare particles. 
     SUMMARY 
     According to an aspect of the present invention, a system for reducing expansion of fluid containing tube and float systems during centrifugation is provided. The system includes at least one chamber and an offset fluid disposed within the at least one chamber. The system includes at least one tube and float system that contains a suspension suspected of having a target material. The at least one tube and float system is disposed within the at least one chamber. The system also includes a centrifuge for centrifugally processing the suspension disposed within the at least one tube and float system. The centrifuge centrifugally spins the at least one tube and float system and the at least one chamber in such a manner that within each chamber the offset fluid surrounds at least a part of the tube and the offset fluid acts on the tube to offset forces that expand the volume of the tube. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows an isometric view of an example system for isolating a target material of a suspension. 
         FIG. 2  shows a cross-sectional view along a line A-A, shown in  FIG. 1 , of an assembly including a tube and float system placed within a chamber filled with an offset fluid. 
         FIG. 3  shows a flow diagram summarizing a general method of analyzing a suspension for the presence of a target material. 
     
    
    
     DETAILED DESCRIPTION 
     This disclosure is directed to systems and methods for isolating a target material of a suspension. A suspension is a fluid containing particles that are sufficiently large for sedimentation. Examples of suspensions include paint, urine, anticoagulated whole blood, and other bodily fluids. A target material can be cells or particles whose density equilibrates when the suspension is centrifuged. Examples of target materials found in suspensions obtained from living organisms include cancer cells, ova, inflammatory cells, viruses, parasites, and microorganisms, each of which has an associated specific gravity. The system for isolating a target material includes at least one tube and float system, at least one chamber, an offset fluid disposed within each chamber, and a centrifuge for centrifugally processing the suspension disposed within the at least one tube disposed within the at least one chamber. 
       FIG. 1  shows an isometric view of an example system  100  for isolating a target material of a suspension. In the example of  FIG. 1 , the system  100  includes four chambers  101 - 104  attached to four slots in a rotor  105 , two tube and float systems  106  and  108  are disposed within the chambers  101  and  103 , and a centrifuge  109 . Each of the chambers  101 - 104  includes an offset fluid (not shown). Systems for isolating a target material are not limited to just four chambers and two tube and float systems. In other embodiments, the number of chambers can be greater or less than four and each chamber can be dimensioned to include at least one tube and float system. 
       FIG. 2  shows a cross-sectional view along a line A-A, shown in  FIG. 1 , of an assembly  100  including the tube and float system  108  placed within a chamber  103  filled with an offset fluid  135 . The system  108  includes a tube  130  and a float  110 , which is shown suspended within a suspension  111 . The tube  130  can have a circular cross-section, a first closed end  132 , and a second open end  134 . The open end  134  is sized to receive a stopper or cap  140 , but the open end  134  can also be configured with threads (not shown) to receive a threaded stopper or screw cap  140  that can be screwed onto the open end  134 . Other closure means are also contemplated, such as parafilm. The tube  130  can also be configured with two open ends that are both sized and configured to receive stoppers or caps. As shown in the example of  FIG. 2 , the tube  130  has a generally cylindrical geometry, but may also be configured with a tapered geometry that widens toward the open end  134 . Although the tube  134  has a circular cross-section, in other embodiments, the tube  134  can have an elliptical, a square, a rectangular, an octagonal, or any other suitable cross-sectional shape that substantially extends the length of the tube. 
     The tube  130  is formed of a transparent or semi-transparent material and the sidewall  136  of the tube  130  is sufficiently flexible or deformable such that it expands in the radial direction during centrifugation, e.g., due to the resultant hydrostatic pressure of the sample under centrifugal load. As the centrifugal force is removed, the tube sidewall  136  substantially returns to its original size and shape. 
     The tube  130  may be formed of any transparent or semi-transparent, flexible material (organic and inorganic), such as polystyrene, polycarbonate, styrene-butadiene-styrene (“SBS”), styrenelbutadiene copolymer, such as K-Resin®. However, the tube  130  does not necessarily have to be clear, as long as the receiving instrument examining the tube  130  for the target material of the suspension can capture images or detect the target material in the tube  130 . For example, target materials with a very low level of radioactivity that cannot be detected in a suspension through a non-clear or semi-transparent wall  136  after it is separated by the process of the present invention and trapped between the tube wall  136  and the float  110  as described below. 
     A variety of different floats can be used for various different analyses, and the present invention is not limited to any particular float. In the example shown in  FIG. 2 , the float  110  includes a main body portion  112 . The float  110  can he composed of one or more generally rigid organic or inorganic materials, such as a rigid plastic material, such as polyoxymethylene (“Delrin®”), polystyrene, 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, and so forth, and most preferably polystyrene, polycarbonate, polypropylene, acrylonitrile butadiene-styrene copolymer (“ABS”) and others. 
     In this regard, one of the objectives of the present invention is to avoid the use of materials and/or additives that interfere with the detection or scanning method. For example, if fluorescence is utilized for detection purposes, the material utilized to construct the float  110  does not create “background” fluorescence at the wavelength of interest. 
     The main body portion  112  of the float  110  is sized to have an outer diameter  118  that is less than the inner diameter  138  of the tube  130 . The difference in float outer diameter  118  and the tube inner diameter  138  defines an annular channel or gap  150  between the float  110  outer surface and the inner sidewall  136  of the tube  130 . The main body portion  112  occupies much of the cross-sectional area of the tube  130 , the annular gap  150  being large enough to contain the target material of the suspension  111 . 
     When the tube and float system  100  is centrifuged, the tube expands, causing the annular gap  150  to increase. As centrifugation is slowed, the annular gap  150  decreases in size returning to its static dimension. As the tube  130  contracts, however, pressure may build up in the fluid located below the float  110 . This pressure may cause particles located within the fluid below the float  110  to be forced into the annular gap  150  which contains the target material, thus making imaging or detecting the target material in the annular gap  150  more difficult. Alternatively, the collapse of the side wall  136  of the tube  130  during deceleration may produce excessive or disruptive fluid flow through the expanded layers located within the annular gap  150 . 
     To counteract the radial expansion forces, represented by directional arrows  131 , acting on the tube  130 , the chamber  103  is at least partially filled with the offset fluid  135 . In certain embodiments, as shown in the example of  FIG. 2 , the offset fluid  135  can be filled to approximately the same level as the suspension  111  in the tube  130 . In other embodiments, the offset fluid  135  can be filled to a level that is less than the level of the suspension  111  in the tube  130 . In still other embodiments, the offset fluid  135  can be filled to a level that is greater than the level of the suspension  111  in the tube  130 . In certain embodiments, the offset fluid  135  can be any fluid or gel that has a density substantially equal to the density of the suspension. For example, the offset fluid  135  can be water, oil, silica gel, silica oil, or a saline solution, etc. In other embodiments, the offset fluid  135  has density that is lower than the density of the suspension  111 . In still other embodiments, the offset fluid  135  has a density that is greater than the density of the suspension  111 . As explained below, during centrifuging, the offset fluid  135  exerts forces  137  that are directed radially inward on the tube  130  offsetting the outward radial forces  131 . The forces  137  acting inwardly against the tube  130  are approximately equal to the outward acting forces  131  from the suspension  111  contained within the tube  130  at any point on the tube  130  from the center of rotation. 
     The appropriate overall specific gravity of the float  110  depends on the application. Suppose, for example, the suspension  111  is a whole blood sample. The specific gravity of the float  110  can be selected with a specific gravity between that of red blood cells (approximately 1.090) and that of plasma (approximately 1.028). The float  110  may be formed of multiple materials having different specific gravities, so long as the composite specific gravity of the float is within the desired range. The specific gravity of the float  110  and the volume of the annular gap  150  may be selected so that some red cells and/or plasma is retained near the ends of the annular gap  150 , as well as the bully coat layers. Upon centrifuging, the float  110  occupies the same axial position as the buffy coat layers and target cells. For example, the float  110  can rest on the packed red cell layer and the buffy coat is retained in the narrow annular gap  150  between the float  110  and the inner wall  136  of the tube  130 . The expanded buffy coat region can then be examined under illumination and magnification or imaged to identify circulating epithelial cancer or tumor cells or other target materials. 
     In one embodiment, the density of the float  110  can be selected so that the float  110  is located within the granulocyte layer of the centrifuged blood sample. The granulocytes are located within the buffy coat layer above the packed red-cell layer and have a specific gravity of about 1.08-1.09. In this embodiment, the float  110  is selected with a specific gravity in the range of about 1.08 to about 1.09 such that, upon centrifugation, the float  110  is located within the granulocyte layer. The amount of granulocytes can vary from patient to patient by as much as a factor of about twenty. Therefore, selecting the float specific gravity to substantially match the specific gravity of the granulocyte layer is advantageous because loss of any of the lymphocyte/monocyte layers, which are located above the granulocyte layer, is avoided. During centrifugation, as the granulocyte layer increases in size, the float  110  may be located higher in the granulocyte layer and keep the lymphocytes and monocytes at essentially the same position with respect to the float  110 . 
     In one example method of using the tube and float system  108 , a sample of anticoagulated whole blood is obtained. For example, the whole blood to be analyzed may be drawn using a standard Vacutainer® or other blood collection devices of a type having an anticoagulant predisposed therein. 
     A fluorescently labeled antibody, which is specific to the target epithelial cells or other target materials, can be added to the blood sample and incubated. In one embodiment, the epithelial cells are labeled with anti-epcam having a fluorescent tag attached to it. Anti-epcam binds to an epithelial cell-specific site that is not typically present in other cells normally found in the blood stream. A stain or colorant, such as acridine orange, may also be added to the whole blood sample to cause the various cell types to assume differential coloration for ease of discerning the buffy coat layers under illumination and to highlight or clarify the morphology of epithelial cells during examination of the sample. 
     The float  110  may be placed into the tube  130  before or after the blood sample is introduced into the tube  130 . The tube and float system  108  filled with the labeled whole blood sample is then placed in the chamber  103  containing the offset fluid  135 . When the centrifuging is started, the resultant hydrostatic pressure of the blood sample within the tube  130  and the hydrostatic pressure of the offset fluid  135  offset one another to substantially reduce or prevent radial expansion of the tube wall  136 , which typically occurs in the absence of the offset fluid  135  and chamber  103 . The blood components and the float  110  are free to move under centrifugal motivation within the tube  130 . The blood sample is separated into six distinct layers according to density, which are, from bottom to top: packed red blood cells, reticulocytes, granulocytes, lymphocytes/monocytes, platelets, and plasma. The epithelial cells sought to be imaged tend to also collect in the buffy coat layers, i.e., the granulocyte, lymphocyte/monocyte, and platelet layers as a result of their density. The specific gravity of the float  110  is selected so that it occupies approximately the same axial position as the buffy coat layers. As a result, the contents of the buffy coat are expanded and occupy the narrow annular gap  150 . 
     When the centrifugal separation is complete, and the rotational speed of the tube  130  decreases, the offsetting forces of the blood sample and the offset fluid  135  prevent undesirable fluid flow in the annular region  150  between the float  110  and the tube  130 ; e.g., the type of fluid flow that typically occurs when the tube  130  was allowed to deform. When the centrifugal force is completely removed, the buffy coat layers and/or other target material are disposed within the annular gap  150  for analysis. Optionally, the tube and float system  100  may be transferred to a microscope or optical reader to identify any target materials in the blood sample. 
     Once the buffy coat is separated, the tube  130  may be inspected using an automated inspection system for imaging and analysis. This requires precise positioning of the tube. Therefore, features may be added to the sample tube, e.g., to the bottom of the tube, to facilitate tube engagement, handling, and positioning, e.g., under automated or preprogrammed control. 
     Although system and method embodiments of the present invention have been described for isolating and detecting the presence of target materials in a whole blood sample, embodiments of the present invention are not intended to be so limited.  FIG. 3  shows a flow diagram summarizing a general method of analyzing a suspension for the presence of a target material. In step  301 , a suspension to be analyzed for the presence of a target material is introduced to a tube and float system. In step  302 , the tube and float system containing the suspension are deposited in a chamber containing an offset fluid, as shown in  FIG. 2 . In step  303 , the tube and float system and the chamber including the offset fluid are centrifuged. In step  304 , when the time for centrifuging the tube and float system is complete, the tube and float system are removed from the chamber and contents of the layers located within the annular gap  150  are analyzed. 
     The foregoing description, for purposes of explanation, used specific nomenclature to provide a thorough understanding of the invention. However, it will be apparent to one skilled in the art that the specific details are not required in order to practice the invention. The foregoing descriptions of specific embodiments of the present invention are presented for purposes of illustration and description. They are not intended to be exhaustive of or to limit the invention to the precise forms disclosed. Obviously, many modifications and variations are possible in view of the above teachings. The embodiments are shown and described in order to best explain the principles of the invention and its practical applications, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the following claims and their equivalents: