Abstract:
A tube and float system for use in separation and axial expansion of the buffy coat is provided. The system includes a transparent, or semi-transparent, flexible sample tube and a rigid separator float having a specific gravity intermediate that of red blood cells and plasma. The sample tube has an elongated sidewall having a first cross-sectional inner diameter. The float consists of a main body portion and one or more support members protruding from the main body portion to engage and support the sidewall of the sample tube. The main body portion and the support members of the float have a cross-sectional diameter less than that of the first cross-sectional inner diameter of the tube when the sample tube is expanded, such as by centrifugation. The main body portion of the float together with an axially aligned portion of the sidewall define an annular volume therebetween. The support members protruding from the main body portion of the float traverse said annular volume to produce one or more analysis areas. During centrifugation, the centrifugal force enlarges the diameter of the tube to permit density-based axial movement of the float in the tube. Thereafter, the centrifugal force is reduced to cause the tube sidewall to return to its first diameter, thereby capturing the float and trapping the buffy coat constituents in the analysis area. The bully coat constituents can then be evaluated or measured.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 12/029,274, filed Feb. 11, 2008, now U.S. Pat. No. 7,915,029, which was a continuation of U.S. patent application Ser. No. 11/370,635, filed Mar. 7, 2006, now U.S. Pat. No. 7,329,534, which was itself a divisional of U.S. patent application Ser. No. 10/263,975, filed Oct. 3, 2002, now U.S. Pat. No. 7,074,577. These applications are hereby fully incorporated by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to density-based fluid separation and, in particular, to an improved sample tube and float design for the separation and axial expansion of constituent fluid components layered by centrifugation, and a method employing the same. The present invention finds particular application in blood separation and axial expansion of the buffy coat layers, and will be described with particular reference thereto. However, it will be recognized that the present invention is also amenable to other like applications. 
     BACKGROUND OF THE INVENTION 
     Quantitative Buffy Coat (QBC) analysis is routinely performed in clinical laboratories for the evaluation of whole blood. The buffy coat is a series of thin, light-colored layers of white cells that form between the layer of red cells and the plasma when unclotted blood is centrifuged or allowed to stand. 
     QBC analysis techniques generally employ centrifugation of small capillary tubes containing anticoagulated whole blood, to separate the blood into essentially six layers: (1) packed red cells, (2) reticulocytes, (3) granulocytes, (4) lymphocytes/monocytes, (5) platelets, and (6) plasma. The buffy coat consists of the layers, from top to bottom, of platelets, lymphocytes and granulocytes and reticulocytes. 
     Based on examination of the capillary tube, the length or height of each layer is determined during the QBC analysis and converted into a cell count, thus allowing quantitative measurement of each layer. The length or height of each layer can be measured with a manual reading device, i.e., a magnification eyepiece and a manual pointing device, or photometrically by an automated optical scanning device that finds the layers by measuring light transmittance and fluorescence along the length of the tube. A series of commonly used QBC instruments are manufactured by Becton-Dickinson and Company of Franklin, Lakes, N.J. 
     Since the buffy coat layers are very thin, the buffy coat is often expanded in the capillary tube for more accurate visual or optical measurement by placing a plastic cylinder, or float, into the tube. The float has a density less than that of red blood cells (approximately 1.090 g/ml) and greater than that of plasma (approximately 1.028 g/ml) and occupies nearly all of the cross-sectional area of the tube. The volume-occupying float, therefore, generally rests on the packed red blood cell layer and expands the axial length of the buffy coat layers in the tube for easier and more accurate measurement. 
     There exists a need in the art for an improved sample tube and float system and method for separating blood and/or identifying circulating cancer and/or other rare cells, organisms or particulates or objects (i.e., stem cells, cell fragments, virally-infected cells, trypanosomes, etc.) in the buffy coat or other layers in a blood sample. However, the number of cells expected to be typically present in the buffy coat is very low relative to the volume of blood, for example, in the range of about 1-100 cells per millimeter of blood, thus making the measurement difficult, particularly with the very small sample sizes employed with the conventional QBC capillary tubes and floats. 
     The present invention contemplates a new and improved blood separation assembly and method that overcome the above-referenced problems and others. 
     SUMMARY OF THE INVENTION 
     In a first aspect of the invention, a method of separating and axially expanding the buffy coat constituents in a blood sample includes introducing the blood sample into a flexible sample tube having an elongate sidewall of a first cross-sectional inner diameter. An elongate rigid volume-occupying float is also inserted into, or is present in, the flexible sample tube. 
     The float has a specific gravity intermediate that of red blood cells and plasma. It includes a main body portion and one or more support members protruding from the main body portion of the float to engage and support the sidewall of the sample tube. The main body portion and the support members have a cross-sectional diameter less than the first inner diameter of the tube when the sample tube is subsequently expanded, such as by centrifugation. 
     The main body portion of the float, together with an axially aligned portion of the sidewall of the sample tube, defines an annular volume therebetween. The support members protruding from the main body portion of the float traverse the annular volume to engage and support the sidewall of the tube thereby producing one or more analysis areas. 
     The sample tube containing the blood sample and float is then centrifuged to effect a density-based separation of the blood sample into discrete layers at a rotational speed that causes a resilient expansion or enlargement of the diameter of the sidewall to a second diameter in response to pressure in the blood caused by the centrifugal force, which diameter expansion is sufficiently large to permit axial movement of the float in the tube. During centrifugation, the float is moved into axial alignment with at least the buffy coat layers of the blood sample due to the density of the float. After centrifugation, the rotational speed is reduced and the tube sidewall returns to essentially its first diameter and engages the float. As a result, the buffy coat constituents are trapped in the analysis areas for review, measurement and/or detection by conventional methods. 
     In a further aspect of the invention, an apparatus for separation and analysis of a target analyte in a sample of anticoagulated whole blood is produced. The apparatus includes a transparent, or semi-transparent, flexible tube for holding the sample, the tube having an elongate sidewall of a first cross-sectional inner diameter. The apparatus further includes an elongate, rigid, volume-occupying float having a specific gravity which is intermediate that of red blood cells and plasma. 
     The float includes a main body portion having one or more support members protruding from the main body portion. The cross-sectional diameter of the main body portion and/or the support members of the float are less than the first cross-sectional inner diameter of the tube when the sample tube is subsequently expanded. In this regard, the sidewall is resiliently radially expandable to a second diameter in response to pressure or force. The second diameter is sufficiently large to permit axial movement of the float in the tube during centrifugation. 
     The main body portion of the float, together with an axially aligned portion of the sidewall, defines an annular volume therebetween. The protrusions of the float traverse the annular volume and engage and support the sidewall, forming the analysis area subsequent to centrifugation. 
     In another aspect, a volume occupying separator float adapted for use with an associated sample tube is provided. The float includes a rigid main body portion and one or more support members protruding from the main body portion of the float to engage and support the sidewall of the sample tube. The main body portion and the support members have a cross-sectional diameter less than an inner diameter of the sample tube when the sample tube is expanded. The main body portion together with an axially aligned portion of the sidewall, define an annular volume therebetween. Additionally, the supporting members protruding from the main body portion of the float traverse the annular volume to engage and support the sidewalls and to produce one or more areas for analysis. 
     In a still further aspect, a method for detecting circulating target cells, such as epithelial cancer cells, stem cells, cell fragments, virally-infected cells, trypanosomes, etc., in an anticoagulated whole blood sample is provided. This method includes combining the blood sample with one or more target cell epitope-specific-labeling agents so as to differentiate the target cells from other cells in the blood sample. The blood sample and a volume-occupying separator float are placed into a transparent, or semi-transparent, flexible sample tube. The separator float has a specifically defined specific gravity. It comprises a rigid main body portion and tube support members. The separator float in conjunction with the sidewalls produces one or more areas of analysis. Additionally, the float has a cross-sectional diameter less than an inner diameter of the sample tube when the sample tube is expanded. The blood sample and separator float are centrifuged in the sample tube to effect centrifugally motivated localization of any target cells present in the blood sample to the areas of analysis. The blood sample present in the analysis areas is then examined to identify whether any target cells are present. 
     One advantage of the present invention is found in a blood separating apparatus that can separate the entire buffy coat of a relatively large blood sample from the rest of the blood volume. 
     Another advantage of the invention resides in the fact that the buffy coat layers can be made available for visualization or imaging in one simple operation, i.e., the application of pressure and/or centrifugation. 
     Still another advantage of the invention resides in enhanced buffy coat separation, retention, and, if desired, removal from the sample tube for further processing. 
     Yet another advantage of the invention is found in that the tolerance precision between the float and tube is decreased over that necessary for the prior art QBC-type systems, thus reducing the necessary cost of the components. 
     Still another advantage is found in that the tube can be supported for improved imaging of the sample, and a more repeatable depth for imaging may be provided. 
     Still further advantages of the present invention reside in its relatively simple construction, ease of manufacture, and low cost. 
     In a still additional aspect, the compressibility and/or rigidity of the flexible tube and rigid float can be reversed. In this aspect, the float is designed to shrink in diameter at the higher pressures and moves freely within a rigid, or optionally, semi-rigid tube. The use of a compressible float allows for usage of transparent glass tubes which, in some instances, exhibit enhanced optical properties over polymeric tubes. Furthermore, this aspect generally reduces the tolerance requirements for the glass tubes (since the float would expand up against the tube wall after the pressure decreases), and a full range of float designs is possible. 
     In another aspect, the step of centrifugation is not required. In such an aspect, the application of pressure alone to the inside of the tube, or simply the expansion of the tube (or the compression of the float), is required. For example, such pressure can be produced through the use of a vacuum source on the outside of the tube. Such an application also allows for the top of the sample tube to be kept open and easily accessible. Additionally, the use of a vacuum source may be easier to implement in some situations than the application of a centrifugal force. 
     Additionally, any method of tubular expansion/contraction (or float compression) such as mechanical, electrical, magnetic, etc., can be implemented. Once the tube is expanded (or the float is compressed), the float will move to the proper location due to buoyancy forces created by the density variations within the sample. 
     In a further aspect, the float comprises a part of a collection tube system or assembly. In this aspect, it is not necessary to transfer the sample from a collection container to an analysis tube. The blood or sample fluid can be collected immediately and then tested. Such a system is somewhat faster, and also safer from a biohazard standpoint. For example, this system is desirable in very contagious situations (i.e. Ebola virus, HIV, etc.) where any type of exposure of the blood must be minimized. 
     Still further advantages and benefits of the present invention will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description of the preferred embodiments. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The invention may take form in various components and arrangements of components, and in various steps and arrangements of steps. The drawings, in which like reference numerals denote like components throughout the several views, are only for purposes of illustrating various embodiments of the invention and are not to be construed as limiting the invention. 
         FIG. 1  is a sectional view of a sample tube containing a generally spool-shaped separator float according to an exemplary embodiment of the invention. 
         FIG. 2  is an elevational view of a separator float having generally conical ends according to another exemplary embodiment of the invention. 
         FIG. 3  is an elevational view of a separator float having generally frustoconical ends according to another exemplary embodiment of the invention. 
         FIG. 4  is an elevational view of a separator float according to yet another exemplary embodiment, wherein the ends are generally convex or dome shaped. 
         FIG. 5  is an elevational view of a separator float according to still another exemplary embodiment having sealing ridges offset from the ends. 
         FIGS. 6-8  are elevational views of ribbed separator floats according to further exemplary embodiments of the invention. 
         FIG. 9  is an elevational view of a separator float according to another exemplary embodiment of the present invention having generally helical tube support ridges. 
         FIG. 10  is an elevational view of a separator float according to a further embodiment of the invention having support ribs, which are tapered in the radial direction. 
         FIG. 11  is an elevational view of a separator float according to yet another exemplary embodiment of the present invention having generally tapered, helical tube support ridges. 
         FIG. 12  is an elevational view of a separator float according to another embodiment of the invention having support ribs, which are rounded in cross-sectional shape. 
         FIG. 13  is an elevational view of a separator float according to another embodiment of the invention having helical support ridges, which are rounded in cross-sectional shape. 
         FIG. 14  is an elevational view of a splined separator float according to another exemplary embodiment of the invention. 
         FIG. 15  is an enlarged cross-sectional view taken along the lines  15 - 15  shown in  FIG. 14 . 
         FIG. 16  is an elevational view of a further splined separator float embodiment of the invention. 
         FIGS. 17 and 18  are elevational views of additional splined float embodiments in accordance the invention. 
         FIG. 19  is a perspective view of yet another splined float embodiment of the present invention. 
         FIG. 20  is a perspective view of a float of still another exemplary embodiment wherein the support ridges include intersecting annular ribs and splines. 
         FIGS. 21-26  are elevational views of knobbed or studded separator floats having generally rounded protrusions in various configurations, in accordance with further exemplary embodiments of the present invention. 
         FIGS. 27 and 28  are elevational views of spiked or studded separator floats having facet-like protrusions according to additional exemplary embodiments of the invention. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Turning now to the drawings, wherein the showings are for purposes of illustrating the preferred embodiments of the invention only and not for limiting the same,  FIG. 1  shows a blood separation tube and float assembly  100 , including a sample tube  130  having a separator float or bobber  110  of the invention therein. 
     The sample tube  130  is generally cylindrical in the depicted embodiment, although tubes having polygonal and other geometrical cross-sectional shapes are also contemplated. The sample tube  130  includes a first, closed end  132  and a second, open end  134  receiving a stopper or cap  140 . Other closure means are also contemplated, such as parafilm or the like. In alternative embodiments, not shown, the sample tube may be open at each end, with each end receiving an appropriate closure device. 
     Although the tube is depicted as generally cylindrical, the tube  130  may be minimally tapered, slightly enlarging toward the open end  134 , particularly when manufactured by an injection molding process. This taper or draft angle is generally necessary for ease of removal of the tube from the injection molding tool. 
     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 may be formed of any transparent or semi-transparent, flexible material (organic and inorganic), such as polystyrene, polycarbonate, styrene-butadiene-styrene (“BBB”), styrene/butadiene copolymer (such as “K-Resin®” available from Phillips 66 Co., Bartlesville, Okla.), etc. Preferably, the tube material is transparent. However, the tube does not necessarily have to be clear, as long as the receiving instrument that is looking for the cells or items of interest in the sample specimen can “see” or detect those items in the tube. For example, items of very low level of radioactivity that can&#39;t be detected in a bulk sample can be detected through a non-clear or semi-transparent wall after it is separated by the process of the present invention and trapped near the wall by the float  110  as described in more detail below. 
     In a preferred embodiment, the tube  130  is sized to accommodate the float  110  plus at least about five milliliters of blood or sample fluid, more preferably at least about eight milliliters of blood or fluid, and most preferably at least about ten milliliters of blood or fluid. In an especially preferred embodiment, the tube  130  has an inner diameter  138  of about 1.5 cm and accommodates at least about ten milliliters of blood in addition to the float  110 . 
     The float  110  includes a main body portion  112  and two sealing rings or flanges  114 , disposed at opposite axial ends of the float  110 . The float  110  is formed of one or more generally rigid organic or inorganic materials, preferably a rigid plastic material, such as 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, and so forth, and most preferably polystyrene, polycarbonate, polypropylene, acrylonitrite 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  must not have interfering or “background” fluorescence at the wavelength of interest. 
     The main body portion  112  and the sealing rings or support members  114  of the float  110  are sized to have an outer diameter  118  which is less than the inner diameter  138  of the sample tube  130 , under pressure or centrifugation. The main body portion  112  of the float  110  is also less than the sealing or support rings  114 , thereby defining an annular channel  150  between the float  110  and the sidewall  136  of the tube  130 . The main body portion occupies much of the cross-sectional area of the tube, the annular gap  150  being large enough to contain the cellular components of the buffy coat layers and associated target cells when the tube is the non-flexed state. Preferably, the dimensions  118  and  138  are such that the annular gap  150  has a radial thickness ranging from about 25-250 microns, most preferably about 50 microns. 
     While in some instances the outer diameter  118  of the main body portion  112  of the float  110  may be less than the inner diameter  138  of the tube  130 , this relationship is not required. This is because once the tube  130  is centrifuged (or pressurized), the tube  130  expands and the float  110  moves freely. Once the centrifugation (or pressurization) step is completed, the tube  130  constricts back down on the sealing rings or support ridges  114 . The annular gap or channel  150  is then created, and sized by the height of the support ridges or sealing rings  114  (i.e., the depth of the “pool” is equal to the height of the support ridges  114 , independent of what the tube diameter is/was). 
     In an especially preferred embodiment, the float dimensions are 3.5 cm tall×1.5 cm in diameter, with a main body portion sized to provide a 50-micron gap for capturing the buffy coat layers of the blood. Thus, the volume available for the capture of the buffy coat layer is approximately 0.08 milliliter. Since the entire buffy coat layer is generally less than about 0.5% of the total blood sample, the preferred float accommodates the entire quantity of buffy layer separated in an eight to ten milliliter sample of blood. 
     The sealing or support flanged ends  114  are sized to be roughly equal to, or slightly greater than, the inner diameter  138  of the tube. The float  110 , being generally rigid, can also provide support to the flexible tube wall  136 . Furthermore, the large diameter portions  114  provide a sealing function to maintain separation of the blood constituent layers. The seal formed between the large diameter regions  114  of the float and the wall  136  of the tube may form a fluid-tight seal. As used herein, the term “seal” is also intended to encompass near-zero clearance or slight interference between the flanges  114  and the tube wall  136  providing a substantial seal which is in most cases, adequate for purposes of the invention. 
     The sealing rings  114  are most preferably continuous ridges, in which case the sample may be centrifuged at lower speeds and slumping of the separated layers is inhibited. However, in alternative embodiments, the sealing ridges can be discontinuous or segmented bands having one or openings providing a fluid path in and out of the annular gap  150 . The sealing ridges  114  may be separately formed and attached to the main body portion  112 . Preferably, however, the sealing ridges  114  and the main body portion  112  form a unitary or integral structure. 
     The overall specific gravity of the separator float  110  should be between that of red blood cells (approximately 1.090) and that of plasma (approximately 1.028). In a preferred embodiment, the specific gravity is in the range of from about 1.089-1.029, more preferably from about 1.070 to about 1.040, and most preferably about 1.05. 
     The float may be formed of multiple materials having different specific gravities, so long as the overall specific gravity of the float is within the desired range. The overall 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 may be retained within the annular gap, as well as the buffy coat layers. Upon centrifuging, the float  110  occupies the same axial position as the buffy coat layers and target cells and floats on the packed red cell layer. 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, to identify circulating epithelial cancer or tumor cells or other target analytes. 
     In one preferred embodiment, the density of the float  110  is selected to settle in the granulocyte layer of the blood sample. The granulocytes settle on, or just above, the packed red-cell layer and have a specific gravity of about 1.08-1.09. In this preferred embodiment, the specific gravity of the float is in this range of from about 1.08 to about 1.09 such that, upon centrifugation, the float settles in 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 density such that the float settles in the granulocyte layer is especially advantageous since loss of any of the lymphocyte/monocyte layer, which settles just above the granulocyte layer, is avoided. During centrifugation, as the granulocyte layer increases in size, the float settles higher in the granulocytes and keeps the lymphocytes and monocytes at essentially the same position with respect to the float. 
     The method for detecting circulating epithelial cancer cells in a blood of a subject is disclosed in U.S. Pat. No. 6,197,523 may advantageously be modified to employ the sample tube and float system of the subject invention. The aforementioned U.S. Pat. No. 6,197,523 is incorporated herein by reference in its entirety. 
     In a preferred exemplary method of using the tube/float system  100  of the invention, a sample of anticoagulated blood is provided. For example, the blood to be analyzed may be drawn using a standard Vacutainer® or other like blood collection device of a type having an anticoagulant predisposed therein. 
     A fluorescently labeled antibody, which is specific to the target epithelial cells or other target analytes of interest, can be added to the blood sample and incubated. In an exemplary 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 expected to be present in any other cell normally found in the blood stream. A stain or colorant, such as acridine orange, may also be added to the 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 blood is then transferred to the assembly  100  for centrifugation. The float  110  may be fitted into the tube  130  after the blood sample is introduced into the sample tube  130  or otherwise may be placed therein beforehand. The tube and float assembly  100  containing the sample is then centrifuged. Operations required for centrifuging the blood by means of the subject tube/float system  100  are not expressly different from the conventional case, although, as stated above, reduced centrifuge speeds may be possible and problems of slumping may be reduced. An adaptor may optionally be utilized in the rotor to prevent failure of the flexible tube due to stress. 
     When the centrifugation is started, the resultant hydrostatic pressure deforms or flexes the wall  136  so as to enlarge the diameter of the tube. The blood components and the float  110  are thus free to move under centrifugal force 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 collect by density in the buffy coat layers, i.e., in the granulocyte, lymphocyte/monocyte, and platelet layers. Due to the density of the float, it occupies the same axial position as the buffy coat layers which thus occupy the narrow annular gap  150 , potentially along with a small amount of the red cell and/or plasma). 
     After centrifugal separation is complete and the centrifugal force is removed, the tube  130  returns to its original diameter to capture or retain the buffy coat layers and target analytes within the annular gap  150 . The tube/float system  100  is transferred to a microscope or optical reader to identify any target analytes in the blood sample. 
       FIGS. 2-28  illustrate several exemplary modifications of the float according to the invention.  FIG. 2  illustrates a float  210  that is similar to the float  110  shown and described by way of reference to the of  FIG. 1 , which includes a main body portion  212  and sealing rings  214 , but which further including a tapered or cone-shaped endcap member  216  disposed at each end. The tapered endcaps  216  are provided to facilitate and direct the flow of cells past the float  210  and sealing ridges  214  during centrifugation. 
       FIG. 3  illustrates a float  310 , which is similar to the float  210  shown and described by way of reference to  FIG. 2 , including a main body portion  312  and sealing ridges  314 , but having truncated cone-shaped endcap members  316 , disposed at each end. The frustoconical endcaps  316  are provided to facilitate the movement or flow of cells and the float during centrifugation. 
       FIG. 4  illustrates a float  410 , which is substantially as shown and described by way of reference to the floats  210  and  310  of  FIGS. 2 and 3 , respectively, but where instead, generally convex or dome-shaped members  416 , which cap the sealing ridges  414 . The endcaps  416  may be hemispherical, hemiellipsoidal, or otherwise similarly sloped, are provided. Again, the sloping ends  416  are provided to facilitate density-motivated cell and float movement during centrifugation. 
     The geometrical configurations of the endcap units  216 ,  316 , and  416  illustrated in  FIGS. 2-4 , respectively, are intended to be exemplary and illustrative only, and many other geometrical shapes (including concave or convex configurations) providing a curved, sloping, and/or tapered surface around which the blood sample may flow during centrifugation. Additional exemplary shapes contemplated include, but are not limited to tectiform and truncated tectiform; three, four, or more sided pyramidal and truncated pyramidal, ogival or truncated ogival; geodesic shapes, and the like. 
       FIG. 5  illustrates a float  510  similar to the embodiment depicted in  FIG. 1 , but wherein the sealing ridges are  514  are axially displaced from the ends. Optional endcap members  516  appear as conical in the illustrated embodiment. However, it will be recognized that the endcaps  516 , if present, any other geometrical configuration which provides a sloped or tapered surface may be used, as described above. 
     Although the remaining  FIGS. 6-28  are illustrated with generally flat ends, i.e., without tapered ends, it will be recognized that each of the illustrated embodiments may optionally be modified to include any of the end cap types shown above in  FIGS. 2-5 , or other geometrical configuration which provide a sloped or tapered surface. 
       FIGS. 6-13  illustrate embodiments of the invention having generally annular tube support members.  FIG. 6  illustrates a ribbed float  610  having a plurality of annular ribs or ridges  620  axially spaced along a central body portion  612 . Optional end sealing ridges  614  are disposed at opposite ends of the float. The ribs  620  and the optional end sealing ridges  614  are sized to provide a sealing engagement with the tube  130  ( FIG. 1 ) when a centrifugal force is removed. The flexible tube expands during centrifugation to permit flow therearound during the density-based centrifugal separation process. The main body portion  612  has a diameter smaller than the inner diameter of the tube during centrifugation and while supported by rib  614  and, thus, multiple annular channels  650  are defined between the main body portion  612  and the inner tube wall upon completion of the centrifugation process. 
     Although the illustrated embodiment in  FIG. 6  depicts continuous ribs, it will be recognized that the support ribs may likewise be broken or segmented to provide an enhanced flow path between adjacent annular channels  650 . Additionally, multiple ribs and/or sealing ridges may be present in order to provide support for the deformable tube and/or to prevent the tube walls from collapsing inwardly. 
       FIG. 7  illustrates a float  710  according to a further embodiment. The float  710  is similar to the float  610  shown in  FIG. 6 , and has a plurality of ribs  720  axially spaced along a central body portion  712 , and wherein plural annular channels  750  are defined therebetween as described above, but wherein the tube support ribs  720  are less densely spaced apart than in the  FIG. 6  embodiment. Optional sealing ridges  714  are disposed at opposite ends of the float. Again, the illustrated embodiment depicts continuous ribs, however, it will be recognized that the support ribs may likewise be broken or segmented to provide an enhanced flow path between adjacent annular channels  750 . 
       FIG. 8  illustrates a further float embodiment  810 , similar to the embodiments of  FIGS. 6 and 7 , the above descriptions of which are equally applicable thereto. However, the float  810  differs in that it lacks sealing ridges at the opposite ends thereof, which may optionally be provided, and the spacing of the ribs  820  is intermediate the rib spacing shown in  FIGS. 6 and 7 . 
       FIG. 9  illustrates a further float embodiment  910 , wherein a helical support member or ridge  920  is provided. That is, instead of discrete annular bands, multiple turns of the helical ridge  920  provides a series of spaced apart ridges on the main body portion  912 , which defines a corresponding helical channel  950 . The helical ridge  920  is illustrated as continuous, however, the helical band may instead be segmented or broken into two or more segments, e.g., to provide path for fluid flow between adjacent turns of the helical buffy coat retention channel  950 . Optional sealing ridges  914  appear at each axial end of the float  910 . 
       FIGS. 10 and 11  illustrate further ribbed and helical float embodiments  1010  and  1110 , respectively. In  FIG. 10 , annular support ribs  1020 , on a main body portion  1012 , are tapered in the radial dimension. In  FIG. 11 , a tapered helical support  1120  appears, formed on a main body portion  1112 . The floats  1010  and  1110  are otherwise as described above by way of reference to  FIGS. 6 and 9 , respectively. Although the support members  1020  and  1120  are shown as continuous, they may alternatively be discontinuous or segmented to facilitate axial flow. Option sealing ridges, as described above, at opposite axial ends of the floats  1010  and  1110  are omitted in the illustrated embodiment, and may optionally be provided. 
       FIGS. 12 and 13  illustrate still further ribbed and helical float embodiments  1210  and  1310 , respectively. Appearing are support members  1220  and  1320 , formed on respective main body portions  1212  and  1312 . The tube support members  1220  and  1320  each have a generally curved or rounded cross-sectional profile. The floats  1210  and  1310  are otherwise as described above by way of reference to  FIGS. 6 and 9 , respectively. Again, the support members  1220  and  1320  are shown as continuous but may, in alternative embodiments, be discontinuous or segmented. Optional end sealing ridges  1314  appear in  FIG. 13 . Furthermore, end sealing ridges do not appear in  FIG. 12 , but may optionally be provided. 
     Referring now to  FIGS. 14 and 15 , there is shown a splined separator float  1410 . The float  1410  includes a plurality of axially-oriented splines or ridges  1424  radially spaced about a central body portion  1412 . Optional end sealing ridges  1414  are disposed at opposite ends of the float. The splines  1424  and the optional end sealing ridges  1414  protrude from the main body  1412  to engage and provide support for the deformable tube. Where provided, the end sealing ridges  1414  provide a sealing function as described above. The axial protrusions  1424  define fluid retention channels  1450 , between the tube inner wall and the main body portion  1412 . The surfaces  1413  of the main body portion disposed between the protrusions  1424  may be curved, e.g., when the main body portion is cylindrical, however, flat surfaces  1413  are also contemplated. Although the illustrated embodiment depicts splines  1424  that are continuous along the entire axial length of the float, segmented or discontinuous splines are also contemplated. 
       FIG. 16  illustrates a further splined float embodiment  1610  similar to the float  1410  as shown and described above by way of reference to  FIGS. 14 and 15 , but wherein optional end sealing ridges are not provided. 
       FIGS. 17 and 18  are elevational views of alternative splined floats  1710  and  1810 , respectively, and are similar to the respective embodiments shown and described above by way of reference to respective  FIGS. 14 and 16 , but wherein the axial splines  1724  and  1824 , respectively, protruding from respective main body portions  1712  and  1812  are more sparsely radially spaced. The float  1710  includes optional end sealing ridges  1714 ; such do not appear on the float  1810  of  FIG. 18 . As above, the respective surfaces  1713  and  1813  may be flat or curved. 
     Referring now to  FIG. 19 , there is shown a perspective view of a splined separator float  1910  in accordance with a further embodiment of the invention. Multiple axially oriented splines  1924  are spaced radially about and protrude from a central body portion  1912  to provide support for the flexible tube. Optional sealing end ridges  1914  are disposed at opposite ends of the float  1910 . Fluid retention channels  1950  formed between adjacent splines  1924  are defined by adjacent splines  1924  and surfaces  1913  on the main body portion  1912 . The surfaces  1913  are depicted as generally flat, although curved surfaces are also contemplated. The axial splines  1924  are depicted as continuous along the length of the tube; however, segmented or discontinuous splines are also contemplated. 
     Referring now to  FIG. 20 , there is shown yet another embodiment  2010 , including a tube supporting member  2026  protruding with respect to a main body portion  2012 . The support means  2026  can be described as an intersecting network of annular rings or ribs  2020  and axial splines  2024 . Optional end sealing ridges  2014  are disposed at opposite ends of the float. The support member  2026  and the optional sealing ridges  2014  radially protrude from the main body portion  2012  at opposite ends of the float to engage and provide support for the deformable tube. Where provided, the end sealing ridges  2014  provide a sealing function as described above. The raised support member  2026  defines a plurality of fluid retention windows  2050  formed between the tube inner wall and the main body portion  2012 . Surfaces  2013  of the main body portion  2012  corresponding to the windows  2050  may be curved, e.g., when the main body portion is cylindrical, however, flat surfaces  2013  are also contemplated. Although the illustrated embodiment depicts the support member  2026  as a network of annular ribs and axial splines which is continuous, breaks may also be includes in the annular and/or axial portions of the network  2026 , e.g., to provide a fluid path between two or more of the windows  2050 . 
       FIGS. 21-26  illustrate several floats having a plurality of protrusions thereon for providing support for the deformable walls of the sample tube. Referring to  FIGS. 21 and 22 , float  2110  and  2210 , respectively, include multiple rounded bumps or knobs  2128  spaced over the surface of a central body portion  2112 . Optional end sealing ridges  2114  ( FIG. 21 ) are disposed at opposite ends of the float  2110  and do not appear on the float  2210  of  FIG. 22 . The knobs  2128  and the optional end sealing ridges  2114  radially protrude from the main body  2112  and traverse an annular gap  2150  to engage and provide support for the deformable tube wall. Where provided, the end sealing ridges  2114  provide a sealing function as described above. The surface of the main body portion disposed between the protrusions may be curved, e.g., when the main body portion is cylindrical, or, alternatively, may have flat portions or facets. 
     In  FIGS. 23 and 24 , there are illustrated float embodiments  2310  and  2410 , which are as substantially as described above by way of reference to  FIGS. 21 and 22 , respectfully, but wherein the protrusions  2328  form an aligned rather than staggered pattern over the surface of the main body portion  2312 . Optional end sealing ridges  2314  appear in the  FIG. 23  embodiment. 
     Referring now to  FIGS. 25 and 26 , there are illustrated float embodiments  2510  and  2610 , which are as substantially as described above by way of reference to  FIGS. 21 and 22 , respectfully, but wherein the protrusions  2528  are less densely spaced over the surface of the main body portion  2512 . Optional end sealing ridges  2514  appear in the  FIG. 25  embodiment. 
       FIGS. 27 and 28  illustrate float embodiments  2710  and  2810 , respectively, which include multiple raised facets  2728  spaced over the surface of a central body portion  2712 . Optional end sealing ridges  2714  ( FIG. 27 ) are disposed at opposite ends of the float  2710 , and do not appear in the  FIG. 28  embodiment. The facets  2728  and the optional end sealing ridges  2714  radially protrude from the main body  2712  and traverse an annular gap to engage and provide support for the deformable tube wall and define a plurality of fluid retention windows  2750 . Where provided, the end sealing ridges  2714  provide a sealing function as described above. The surfaces  2713  of the main body portion, disposed between the protrusions  2728  and forming a surface defining the fluid-retention windows  2750 , may be curved surfaces, e.g., when the main body portion is cylindrical. Alternatively, the surfaces  2713  may be flat. In alternative embodiments, the size, spacing density, and alignment patterns of the facets  2718  can be modified extensively. 
     The exemplary embodiments of  FIGS. 21-28  have been described with reference to rounded knobs or square facets as supporting the flexible sample tube, although protrusions of any geometrical configuration may be used. Other geometrical configurations for the protrusions are also contemplated, such as conical or frustoconical spikes, tectiform or truncated tectiform protrusions, cylindrical protrusions, pyramidal or truncated pyramidal protrusions, hemiellipsoidal protrusions, and so forth, as well as any combinations thereof. Likewise, the size, spacing, and pattern of the protrusions can be varied. Where the sample is to be imaged, the size and spacing can be selected in accordance with the imaging field of view and other factors. 
     The invention has been described with reference to the preferred embodiments. Obviously, modifications and alterations will occur to others upon reading and understanding the preceding detailed description. It is intended that the invention be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.