WO2011126867A1

WO2011126867A1 - Buffy coat separator float systems and methods

Info

Publication number: WO2011126867A1
Application number: PCT/US2011/030417
Authority: WO
Inventors: Thomas D. Haubert
Original assignee: Battelle Memorial Institute
Priority date: 2010-03-30
Filing date: 2011-03-30
Publication date: 2011-10-13

Abstract

Tube and float systems for separation and axial expansion of the buffy coat are provided. Generally, the systems include a 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 has 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. During centrifugation, the centrifugal force enlarges the diameter of the tube to permit density-based axial movement of the float in the tube. After centrifugation is ended, the tube sidewall returns to its first diameter, thereby capturing the float and trapping the buffy coat constituents in an annular volume. Several different systems for capturing and retrieving the buffy coat constituents are described.

Description

BUFFY COAT SEPARATOR FLOAT SYSTEMS AND METHODS

BACKGROUND OF THE DISCLOSURE

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 61/318,912, filed on March 30, 2010, and to U.S. Provisional Patent Application Serial No. 61/372,900, filed on August 12, 2010. The disclosure of these applications is hereby fully incorporated by reference.

[0002] The present disclosure relates generally to density-based fluid separation, and in particular to improved sample tubes and float designs for the separation and axial expansion of constituent fluid components layered by centrifugation, and methods employing the same. The present disclosure finds particular application in separation and axial expansion of the buffy coat layers of the blood, and will be described with particular reference thereto.

[0003] 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 mostly white blood cells that form between the layer of red blood cells and the plasma when unclotted blood is centrifuged or allowed to stand.

[0004] 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.

[0005] 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, NJ.

[0006] 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.

[0007] 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.

[0008] The present disclosure contemplates new and improved blood separation assemblies and methods that overcome the above-referenced problems and others.

BRIEF DESCRIPTION

[0009] The present application discloses, in various embodiments, apparatuses and methods for separating and axially expanding the buffy coat constituents in a blood sample. The apparatuses include separator floats and sample tubes.

[0010] Disclosed herein are methods of separating and axially expanding buffy coat constitutents in a blood sample; detecting target cells in a blood sample; and capturing or extracting buffy coat constitutents / target cells in a blood sample. Those methods require introducing the blood sample and a rigid volume-occupying float into a flexible sample tube. The rigid float has a specific gravity intermediate that of red blood cells and plasma, and comprises a main body portion spacedly surrounded radially by the sidewall of the sample tube to form an annular volume therebetween; and one or more support members protruding from the main body portion and engaging the sidewall. The sample tube is centrifuged at a rotational speed that causes enlargement of the sidewall to a diameter sufficiently large to permit axial movement of the float, separation of the blood into discrete layers, and movement of the float into alignment with at least the buffy coat constituents of the blood sample. The rotational speed is reduced to cause the sidewall to capture the float and trap buffy coat constituents in the annular volume, which might be divided into one or more analysis areas.

[0011] Disclosed in some embodiments is a volume-occupying separator float comprising at least a first piece and a second piece. Each piece has a first end, a second end, an exterior surface, and an interior surface. The interior surfaces of the first and second pieces cooperate to form an open passage extending between the first end and the second end. The first and second pieces can be connected together.

[0012] The first and second pieces may have substantially the same three- dimensional shape. In some embodiments, the interior surface of the first piece comprises a semi-cylindrical surface. Put another way, a lateral cross-sectional view of the first piece may have a semi-annular shape. Sometimes, the interior surface of the first piece is substantially planar, i.e. a lateral cross-sectional view of the interior surface of the first piece is substantially a straight line. In some floats, a lateral cross-sectional view of the interior surface of the first piece is substantially a straight line, and a lateral cross-sectional view of the interior surface of the second piece is substantially a straight line with a central indent. In specific embodiments, a lateral cross-sectional view of the open passage may have a rectangular shape or a circular shape.

[0013] The exterior surface of the first and second pieces may each substantially conform to an inner surface of the sidewall of the sample tube in which the two-piece float is used. The exterior surface of the first and second pieces may also each comprise at least one support member for engaging an inner surface of the sidewall.

[0014] The first and second pieces can be joined together using clips, clamps, and other joining mechanisms or devices.

[0015] Sometimes, the first and second piece each further comprise at least one side surface. The first and second pieces can be connected together on the at least one side surface.

[0016] The two-piece or multiple-piece float can be used in conjunction with a flexible bag to capture buffy coat constituents in a blood sample. A blood sample is introduced into a flexible bag, and a float is placed around the flexible bag, then the bag and float are placed in a flexible sample tube. During centrifugation, the float moves into alignment with at least the buffy coat constituents. Upon reducing the rotational speed, the sidewall captures the float. The flexible bag can then be sealed at the first end and the second end of the float to capture the buffy coat constituents. The flexible bag can be sealed by welding the first end and the second end of the float. The welding may be performed ultrasonically. Other sealing and/or enclosure devices or mechanisms are also contemplated.

[0017] Disclosed in other embodiments is a volume-occupying separator float comprising a main body portion. The main body portion has a first end and a second end and at least one pressure seal. A buffy coat passage extends from the second end to the first end and has a centrifugation valve oriented to open during centrifugation, the valve being located at the second end. In some specific embodiments, there is a first pressure seal around the first end and a second pressure seal around the second end of the main body portion. In additional embodiments, a pressure relief passage extends from the second end to the first end and has a pressure relief valve oriented to open when pressure at the second end is greater than pressure at the first end by a specified value. In other embodiments, there is a pressure relief valve but not a buffy coat passage in the separator float.

[0018] During centrifugation, the pressure seal(s) substantially prevent the blood sample from traveling between the float and the inner surface of the sample tube. The buffy coat constituents are trapped in the buffy coat passage, and the float can then be removed from the sample tube to obtain the buffy coat constituents.

[0019] Alternatively, a volume-occupying separator float comprises a main body portion. The main body portion has a first end and a second end. One or more centrifugation valves are circumferentially disposed about the main body portion.

[0020] Disclosed in still other embodiments is a volume-occupying separator float comprising a first piece and a second piece. The first and second pieces cooperate to form a rectangular passage between a first end and a second end of the float, and wherein the first and second pieces are joined at the first end of the float. A slide can be placed or located in the rectangular passage.

[0021] During use, this two-piece float containing a slide is placed in a sample tube and centrifuged. This causes the slide to be coated with the buffy coat constituents of the blood sample. The float can then be removed from the sample tube; and the slide can be extracted from the float. This float can also be used to surround a flexible bag, as described above.

[0022] In other embodiments, a two-piece float comprises a top float and a bottom float. The top float has a density intermediate that of plasma and the buffy coat constituents. The top float comprises (i) a lower support member having an upper surface and a lower surface, and (ii) a pitot tube extending axially from the lower surface of the top float through the upper surface and having a top end located distally from the upper surface. The bottom float has a density intermediate that of the buffy coat constituents and red blood cells. The top float lower support member lower surface and the bottom float are complementarily shaped.

[0023] The two-piece float may be designed to relieve pressure below the bottom float. The top float further comprises a second passage extending from the lower surface to the upper surface of the lower support member. The bottom float comprises a support member having an upper surface and a lower surface. A pressure relief tube extends axially from the lower surface of the bottom float support member through the upper surface and terminates at an upper end. The upper end of the bottom float pressure relief tube extends through the top float second passage.

[0024] The top float may further comprise a manipulator extending axially from the upper surface of the lower support member. The manipulator is used to push the top float towards the bottom float. The manipulator can be considered a handle for handling the top float.

[0025] During centrifugation, the buffy coat constituents become aligned between the top float and the bottom float. The top float is then pushed toward the bottom float to remove the buffy coat constituents through the pitot tube.

[0026] The pitot tube may be integral, or made as a separate piece. When the pitot tube is separate, the top float includes (ii) a first passage from the lower surface of the lower support member to the upper surface of the lower support member. After centrifugation, the pitot tube engages the first passage of the top float, and the top float can then be pushed toward the bottom float to remove the buffy coat constituents through the pitot tube.

[0027] Still other embodiments of a two-piece float comprise a top float and a bottom float. The top float has a density intermediate that of plasma and the buffy coat constituents. The top float comprises (i) a lower lateral support member having an upper surface and a lower surface, and (ii) a manipulator extending axially from the upper surface of the lower lateral support member. The bottom float has a density intermediate that of the buffy coat constituents and red blood cells. The bottom float comprises an upper lateral support member having an upper surface and a lower surface. The top float lower lateral support member and the bottom float upper lateral support member are complementarily shaped to form a recess. The recess can be used to trap the buffy coat constituents, and/or can enclose a slide.

[0028] The bottom float may further comprise a support member extending axially from the lower surface of the upper lateral support member.

[0029] The recess can be substantially formed in the bottom float upper lateral support member, with the top float lower lateral support member covering the recess. Alternatively, the recess can be formed in the top float lower lateral support member and the bottom float upper lateral support member covering the recess.

[0030] During use, the buffy coat constituents become aligned between the top float and the bottom float. The top float is then pushed toward the bottom float to push the buffy coat constituents into the recess, and coat the slide. The two-piece float and the sample tube can be separated, and the buffy coat constituents or the slide can then be removed from the two-piece float.

[0031] In other embodiments, the top float comprises (i) a lower lateral support member having an upper surface and a lower surface, and (ii) a hollow member open on the lower surface of the lower lateral support member and extending axially from the upper surface of the lower lateral support member. The hollow member can be adapted to receive a slide. The bottom float comprises an upper lateral support member for sealing the hollow member.

[0032] Also disclosed in embodiments are additional designs for a volume- occupying separator float. The float comprises a main body portion having a top end and a bottom end. One or more support members protrude laterally from the main body portion. The main body portion and the one or more support members define an annular volume. A pitot tube extends axially from the top end of the main body portion. An internal passage passes through the main body portion and connects the pitot tube to an opening in the annular volume. An upper piece comprises (i) a passageway through which the pitot tube extends, and (ii) a manipulator extending axially away from the main body portion. The upper piece has a lower density than the main body portion. The manipulator acts as a handle for moving the upper piece.

[0033] The upper piece passageway can be located inside the manipulator.

[0034] The float may further comprise a one-way valve in the opening oriented to permit flow from the annular volume into the internal passage. The main body portion internal passage can also connect the pitot tube to a plurality of openings in the annular volume.

[0035] The main body portion may further comprise a pressure relief passage extending from the first end to the second end, the pressure relief passage not intersecting the internal passage.

[0036] In particular embodiments, one support member is located on the bottom end of the main body portion, and the opening connecting to the annular volume is located proximally to the one support member.

[0037] In other embodiments, the float comprises one support member extending laterally from a bottom end of the main body portion and at least one helical support member located proximal to the top end of the main body portion.

[0038] In use, the upper piece is pushed down to force fluid towards the annular volume and push the buffy coat constituents through the pitot tube. These embodiments contemplate the pitot tube being integral with the main body portion.

[0039] Sometimes, the pitot tube is separate from the main body portion. Then, only the main body portion is placed in the tube with the blood sample. Centrifugation causes alignment of the annular volume with at least the buffy coat constituents. The pitot tube is then engaged with the internal passage at the top end of the main body portion. The upper piece, comprising a hollow member that extends axially away from the main body portion and surrounds the pitot tube, is threaded around the pitot tube. The upper piece is then pushed down to force fluid towards the annular volume and push the buffy coat constituents through the pitot tube.

[0040] These and other non-limiting characteristics of the disclosure are more particularly disclosed below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The following is a brief description of the drawings, which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same.

[0042] FIG. 1 is a side view of a sample tube containing a volume-occupying separator float.

[0043] FIG. 2 is a diagram illustrating the methods of the present disclosure. [0044] FIG. 3A is a side cross-sectional view of a sample tube containing a separator float surrounding a flexible bag.

[0045] FIG. 3B is a top cross-sectional view of a first exemplary embodiment of a two-piece float for surrounding a flexible bag.

[0046] FIG. 3C is a top cross-sectional view of a second exemplary embodiment of a two-piece float for surrounding a flexible bag.

[0047] FIG. 3D is a top cross-sectional view of a third exemplary embodiment of a two-piece float for surrounding a flexible bag.

[0048] FIG. 4A is a side cross-sectional view of a first exemplary embodiment of a separator float containing a buffy coat passage for trapping / catching the buffy coat constituents and using a centrifugation valve to control flow through the buffy coat passage.

[0049] FIG. 4B is a side cross-sectional view of a second exemplary embodiment of a separator float containing a buffy coat passage for trapping / catching the buffy coat constituents and using a centrifugation valve to control flow through the buffy coat passage.

[0050] FIG. 4C is a side cross-sectional view of a third exemplary embodiment of a separator float using a centrifugation valve to control flow through an annular volume.

[0051] FIG. 4D is a side cross-sectional view of another exemplary embodiment of a separator float.

[0052] FIG. 5A is a side cross-sectional view of a two-piece separator float containing a slide.

[0053] FIG. 5B is a perspective view of the float of FIG. 5A in a closed position.

[0054] FIG. 5C is a perspective view of the float of FIG. 5A in an open position.

[0055] FIG. 6 is a side cross-sectional view of a two-piece float that traps buffy coat constituents and removes them through a pitot tube.

[0056] FIG. 7A is a side cross-sectional view of a first exemplary embodiment of a two-piece float that traps buffy coat constituents in a recess.

[0057] FIG. 7B is a side cross-sectional view of a second exemplary embodiment of a two-piece float that traps buffy coat constituents in a recess.

[0058] FIG. 7C is a side cross-sectional view of a third exemplary embodiment of a two-piece float that traps buffy coat constituents. [0059] FIG. 8 is a side cross-sectional view of a first exemplary embodiment of a two-piece float for extracting buffy coat constituents,

[0060] FIG. 9 is a side cross-sectional view of a second exemplary embodiment of a two-piece float for extracting buffy coat constituents.

DETAILED DESCRIPTION

[0061] A more complete understanding of the components, processes, and apparatuses disclosed herein can be obtained by reference to the accompanying drawings. These figures are merely schematic representations based on convenience and the ease of demonstrating the present disclosure, and are, therefore, not intended to indicate relative size and dimensions of the devices or components thereof and/or to define or limit the scope of the exemplary embodiments.

[0062] Although specific terms are used in the following description for the sake of clarity, these terms are intended to refer only to the particular structure of the embodiments selected for illustration in the drawings, and are not intended to define or limit the scope of the disclosure. In the drawings and the following description below, it is to be understood that like numeric designations refer to components of like function.

[0063] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). When used in the context of a range, the modifier "about" should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range of "from about 2 to about 10" also discloses the range "from 2 to 10."

[0064] The present disclosure relates generally to apparatuses and assemblies which are useful for separating the various components of a blood sample, based on the density of the various components. Those apparatuses include volume- occupying separator floats, sample tubes, and combinations thereof.

[0065] FIG. 1 is an axial cross-section of a blood separation tube and float assembly 100. The assembly includes a sample tube 110 and a separator float or bobber 130 placed therein. [0066] The sample tube 110 is generally cylindrical. However, sample tubes having polygonal and other geometrical cross-sectional shapes are also contemplated. In other words, the sample tube may have a cross-section that is a polygon having n sides. For example, when n=3, the sample tube has a triangular cross-section. In particular, the sample tube may have a regular polygonal cross- section (i.e. the lengths of each side are substantially equal).

[0067] The sample tube 110 includes a first, closed end 114 and a second, open end 116 receiving a stopper or cap 125. Other closure means are also contemplated, such as parafilm or the like. In alternative embodiments, discussed further herein, the sample tube may be open at each end, with each end receiving an appropriate closure device.

[0068] Although the tube is depicted as generally cylindrical, the tube 110 may be minimally tapered, slightly enlarging toward the open end 116, 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.

[0069] The tube 110 is formed of a transparent or semi-transparent material and the sidewall 112 of the tube 110 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 112 substantially returns to its original size and shape.

[0070] The tube may be formed of any transparent or semi-transparent, flexible polymeric material (organic and inorganic), such as polystyrene, polycarbonate, styrene-butadiene-styrene ("SBS"), styrene/butadiene copolymer (such as "K- Resin®" available from Phillips 66 Co., Bartlesville, Oklahoma), 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 cannot 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 disclosure and trapped near the wall by the float 130 as described in more detail below. Desirably, the sample tube is seamless, at least along those portions of the tube along which the float will travel. [0071] In some embodiments, the tube 110 is sized to accommodate the float 130 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 particular embodiments, the tube 110 has an inner diameter 117 of about 1.5 cm and accommodates at least about ten milliliters of blood in addition to the float 130.

[0072] The float 130 depicted here includes a main body portion 132 and two sealing rings or flanges 140, disposed at opposite axial ends of the float 130. The main body portion 132 and the sealing rings or support members 140 of the float 130 are sized to have an outer diameter which is less than the inner diameter 117 of the sample tube 110, under pressure or centrifugation. Put another way, the outer diameter of the support members is substantially equal to the inner diameter 117 of the sample tube 110 in a non-flexed state, so that the float can be held in a particular location by the sample tube. The main body portion 132 of the float 130 also has a smaller outer diameter 138 which is less than the diameter of the sealing or support rings 140, thereby defining an annular volume 170 between the float 130 and the sidewall 112 of the tube 110. The main body portion occupies much of the cross- sectional area of the tube, with the annular volume 170 being large enough to contain the cellular components of the buffy coat layers (i.e. buffy coat constituents) and associated target cells when the tube is in the non-flexed state. Preferably, the dimensions 138 and 117 are such that the annular volume 170 has a radial thickness ranging from about 25 microns to about 250 microns, most preferably about 50 microns. It should be noted that the term "annular" is used to refer to the ring-like shape formed by the float within the tube, and should not be construed as requiring the shape to be defined by two concentric circles. Rather, the tube and the float may each have different shapes and "annular" refers to the shape formed between them. The number of support members 140 may also vary, as will be seen further herein.

[0073] A bore or channel 150 extends axially through the float 130. When the tube/float system is centrifuged, the tube expands, freeing the float in the blood sample. As centrifugation is slowed, the float is captured by the sidewall 112 of the tube as the sube returns to its original diameter. As the tube continues to contract, pressure may build up in the blood fraction trapped below the float, primarily red blood cells. This pressure may cause red cells to be forced into the annular volume 170 containing the captured buffy coat constituents, thus diluting the contents or making imaging of the contents of the buffy coat more difficult. Alternatively, the collapse of the side wall of the sample tube during deceleration may produce excessive or disruptive fluid flow through the separated buffy coat layers. The bore 150 allows for any excessive fluid flow or any resultant pressure in the dense fractions trapped below the float 130 to be relieved. The excessive fluid flows into the bore 150, thus preventing degradation of the buffy coat sample. This bore can be considered a pressure relief means for inhibiting excessive fluid flow through the buffy coat constituents. The bore is depicted here as being central and axially aligned within the float 130, but other configurations are contemplated so long as the bore extends completely through the float from one end to the other. In some embodiments, the bore 150 is centrally located and axially extending.

[0074] While in some instances the outer diameter 138 of the main body portion 132 of the float 130 may be less than the inner diameter 117 of the tube 110, this relationship is not required. This is because once the tube 110 is centrifuged (or pressurized), the tube 110 expands and the float 130 moves freely. Once the centrifugation (or pressurization) step is completed, the tube 130 constricts back down on the sealing rings or support ridges 140 to capture the float. The annular volume 170 is then created, and sized by the length of the support ridges or sealing rings 140 (i.e., the depth of the "pool" is equal to the length of the support ridges 140, independent of what the tube diameter is/was).

[0075] In desired embodiments, the float dimensions are 3.5 cm tall x 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.

[0076] The sealing or support flanged ends 140 are sized to be substantially equal to, or slightly greater than, the inner diameter 117 of the tube. The float 130, being generally rigid, can also provide support to the flexible tube wall 112. Furthermore, the support members 140 provide a sealing function to maintain separation of the blood constituent layers. The seal formed between the support members 140 of the float and the wall 112 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 140 and the tube wall 112 providing a substantial seal which is, in most cases, adequate for purposes of the disclosure.

[0077] The support members 140 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 which are discussed further herein, the support members can be discontinuous or segmented bands having one or openings providing a fluid path in and out of the annular gap 170. The support members 140 may be separately formed and attached to the main body portion 132. Preferably, however, the support members 140 and the main body portion 132 form a unitary or integral structure.

[0078] The geometrical configuration of the support members are exemplary only, and different configurations are contemplated. For example, the support member 140 in FIG. 1 is flat but support members that are tapered away from the main body portion 132 or concave curved are also contemplated. These shapes can provide a surface that encourages flow of the blood around the float 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.

[0079] The overall specific gravity of the separator float 130 should be between that of red blood cells (approximately 1 .090) and that of plasma (approximately 1.028). In more specific embodiments, the specific gravity is in the range of from about 1.089 to about 1.029, more preferably from about 1.070 to about 1.040, and most preferably about 1 .05.

[0080] 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 130 and the volume of the annular gap 170 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 130 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 170 between the float 130 and the inner wall 112 of the tube 110. 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. [0081] In embodiments, the density of the float 130 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. In other embodiments described further herein, the float may be made from two pieces, and the specific gravity of each piece may differ.

[0082] The float 130 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.

[0083] In this regard, it is desirable 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 130 should not have interfering or "background" fluorescence at the wavelength of interest.

[0084] In some aspects, the compressibility and/or rigidity of the flexible tube and rigid float can be reversed. The float is flexible and designed to shrink in diameter at the higher pressures and moves freely within a 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.

[0085] The method for detecting circulating epithelial cancer cells in a blood of a subject is disclosed in U.S. Patent No. 6,197,523 may advantageously be modified to employ the sample tube and float system of the subject disclosure. The aforementioned U.S. Patent No. 6,197,523 is incorporated herein by reference in its entirety.

[0086] In an exemplary method of using the tube/float system 100 of the disclosure, 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.

[0087] A tag, such as a fluorescently labeled antibody or ligand, which is specific to the target epithelial cells or other target analytes of interest, can be added to the blood sample and incubated prior to centrifugation. 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.

[0088] The blood is then transferred to the assembly 100 for centrifugation. The float 130 may be introduced into the tube 110 after the blood sample is introduced into the sample tube 110 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.

[0089] During centrifugation, the sample tube is spun at a rotational speed sufficient to cause several effects. In particular, the resultant hydrostatic pressure deforms or flexes the wall 112 so as to enlarge the diameter of the tube from a first cross-sectional inner diameter to a second diameter, the second diameter being greater than the first diameter. The second diameter is sufficiently large to permit the blood components and the float 130 to move axially under centrifugal force within the tube 110. The blood sample is separated into six discrete and distinct layers according to density, which are, from bottom to top (most dense to least dense): packed red blood cells, reticulocytes, granulocytes, lymphoc tes/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, the float occupies the same axial position within the sample tube as the buffy coat layers/constituents which thus occupy the narrow annular volume 188, potentially along with a small amount of the red cell and/or plasma). Put another way, the float moves into alignment with at least the buffy coat constituents of the blood sample.

[0090] After centrifugal separation is complete and the centrifugal force is removed, the tube 110 returns to its original diameter to capture or retain the float and the buffy coat layers and target analytes within the annular volume 188. The tube/float system can be transferred to a microscope or optical reader to identify any target analytes in the blood sample. Depending on the subsequent use of the float, the annular volume may be considered to make up one or more analysis areas.

[0091] Centrifugation may not be required. Sometimes 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.

[0092] In additional embodiments described herein, a removal device, such as a syringe, is then used to extract the buffy coat layers / constituents from the annular volume. The intent here is to extract the target cells of interest, so it is acceptable to remove some of the red blood cells and/or plasma during this process as well. If tags have not yet been added, they may be added now to tag or label the "target" cells of interest. Again, the tags are any kind that an analytical instrument or detector could detect, e.g. fluorescent, radioactive, etc. The tags may be in the removal device itself, or they can be added separately.

[0093] The sample is then "squirted" through the instrument / detector and the tagged cells are analyzed. It may be sufficient to count the number of tagged cells. However, in further embodiments, the 'positive' sample cells are diverted into a holder for further analysis. Means of separating such cells are known in the art and can be similar to those used in flow cytometry, for example by coordinating the timing of the instrument / detector with the holder. The positive sample can then be further analyzed, for example by preparing a slide for further examination. This 'squirt-n-divert' method results in a smaller sample volume that is easier to analyze compared to the original blood sample, which was many times larger.

[0094] The float can comprise a part of a collection tube system or assembly. Thus, it is not necessary to transfer the buffy coat 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.

[0095] FIG. 2 is a diagram illustrating the general methods described above. In step 2, the target cells in the buffy coat layers of the blood sample can be tagged prior to centrifugation. In step 4, the buffy coat is isolated, e.g. by centrifugation. In step 6, the sample containing the buffy coat, and reduced in volume compared to the original blood sample, is extracted from the sample tube. In step 8, if the target cells were not already tagged, they can be tagged now. Alternatively, they can be tagged using different tags suitable for use with the given instrument / detector. In step 10, the reduced volume is run through the detector. As illustrated here, the reduced volume with the tagged target cells begin in syringe 20 and are injected into detector 25 which separates the 'positive' sample (i.e. target cells) and diverts them into holder 30. The 'negative' sample goes to waste, i.e. is disposed of. Finally, in step 12, the positive sample is further analyzed.

[0096] The sample tubes, separator floats, and methods described above provide a general idea of the present disclosure. Several further concepts are described herein.

[0097] FIGs. 3A-3D illustrate one concept of a blood separation apparatus 300 where rather than placing the blood sample and float into a sample tube, the blood sample is placed into a flexible bag 302. FIG. 3A is an axial cross-sectional view. Instead of being placed in the bag and contacting the blood sample, the float 320 is placed around the bag 302, i.e. on the exterior of the flexible bag. The flexible bag 302 and the float 320 are then placed into a sample tube 310 and centrifuged. After centrifugation, the buffy coat layers / constituents are in the portion of the bag 302 located between a first end 324 of the float and a second end 326 of the float. The flexible bag 302 is then sealed at the first end 324 and the second end 326 to capture the buffy coat constituents. The sealing may be done, for example, by welding. In particular embodiments, the welding is performed ultrasonically. Ultrasonic welding is an industrial technique commonly used for plastics, whereby high-frequency ultrasonic acoustic vibrations are locally applied to two items being held together to create a solid-state weld between the two items. The term "welding" is used here to indicate the action of closing the bag off in a specific location, and is synonymous with melting.

[0098] Separator floats made from two or more pieces are especially suitable for use with this concept. In embodiments, the separator float 320 comprises a first piece 340 and a second piece 350. The first piece 340 has a first end 341 , a second end 342, an interior surface 344, and an exterior surface 346. In some embodiments, the first piece also includes at least one side surface 348. The second piece 350 has a first end 351 , a second end 352, an interior surface 354, and an exterior surface 356. In some embodiments, the second piece also includes at least one side surface 358. The first piece interior surface 344 and the second piece interior surface 354 cooperate to form an open passage 380 extending between the first end 324 of the float and the second end 326 of the float, or in other words between the first end 341 , 351 and second end 342, 352 of each piece 340, 350. Each piece 340, 350 may also include optional support members 327 on their exterior surface 346, 356 for engaging the sidewall 312. In some embodiments, the exterior surface of each piece 340, 350 comprises at least one support member, and in particular embodiments, the exterior surface of each piece has two support members, i.e. a first support member at the first end and a second support member at the second end.

[0099] In particular embodiments, the first and second pieces have substantially the same three-dimensional shape. FIGs. 3B-3D show lateral cross-sectional views of different first and second pieces. In FIG. 3B, the first piece 340 and second piece 350 are substantially of the same shape. The interior surface 344 of the first piece 340 is substantially planar, i.e. is substantially a straight line in this lateral cross- sectional view. The interior surface 354 of the second piece 350 is also substantially planar. The two interior surfaces are substantially parallel to each other, i.e. the open passage 380 has a rectangular shape. It should be noted that the exterior surface 346, 356 is shown contacting the interior surface 344, 354 at both ends, and that the exterior surface 346, 356 is substantially conforming to an inner surface of the sample tube, for example having a semi-cylindrical surface. In some embodiments, the first piece 340 and/or second piece 350 may have at least one side surface 348, 358 (shown here as a dotted line). In such embodiments having a side surface, the exterior surface may be described as an arcuate surface, or in a lateral cross-sectional view the exterior surface is an arc. The side surface 348, 358 is generally perpendicular to the interior surface 344, 354.

[0100] The first piece 340 and second piece 350 can be connected together using any means. For example, in FIG. 3B, the first piece and second piece are joined on one side by a hinge mechanism 374, and on the other side by clips 375. Other connecting mechanisms, such as tongue-and-groove, detent-and-catch, hook-and- loop, etc., can also be used. The connecting mechanism can be located on the interior surface or a side surface.

[0101] In FIG. 3C, the interior surface 344 of the first piece 340 is substantially planar. The second piece 350 has a semi-annular cross-sectional shape. Put another way, the interior surface 354 is substantially a straight line with a central indent. Put yet another way, the interior surface 354 of the second piece comprises a semi-cylindrical surface 355. [0102] In FIG. 3D, the first piece 340 and the second piece 350 each have a semi-annular cross-sectional shape. The open passage 380 has a circular shape.

[0103] FIG. 4A shows another concept of a blood separation apparatus 400 including a sample tube 410 and a separator float 420. The sample tube 410 is formed from a sidewall 412 and has a first, closed end 414 and a second, open end 416. The separator float 420 includes a main body portion 422 having a first end 424 and a second end 426. A pressure relief passage 487 extends from the second end 426 to the first end 424, and has a first one-way pressure relief valve 434 oriented to open when pressure at the second end 426 is greater than pressure at the first end 424 by a specified value. Put another way, the first pressure relief valve

434 does not open until there is a specified difference between the pressure at the second end 426 and the pressure at the first end 424. A buffy coat passage 478 extends from the second end 426 to the first end 424 and has a centrifugation valve

435 oriented to open during centrifugation. At least one pressure seal wraps around the main body portion. Two pressure seals 404 are shown here, one extending radially from the first end 424 and the other from the second end. The pressure seals effectively prevent fluid flow between the main body portion 422 and the sidewall 412 of the sample tube.

[0104] When the apparatus of FIG. 4A is used, during centrifugation, the centrifugation valve 435 opens as necessary to allow the blood sample to separate into discrete layers. The centrifugation valve 435 may be thought of as a weight on a spring. When exposed to higher forces during centrifugation, the valve opens and allows red blood cells, plasma, and buffy coat constituents to flow through the buffy coat passage 478. As the centrifuge begins to slow down, the valve closes to seal the buffy coat passage. After centrifugation, buffy coat constituents reside in the buffy coat passage 478. Pressure built up underneath the float 420 can be relieved through the pressure relief passage 487 with minimal disturbance to the buffy coat constituents in the buffy coat passage 478. The float 420 can be removed from the sample tube 410 with the buffy coat constituents being present in the buffy coat passage 478.

[0105] FIG. 4B depicts an apparatus similar to the apparatus of FIG. 4A, except that there is no pressure relief passage 487. It is contemplated here that in the event of a pressure difference between the first end 424 and the second end 426, the pressure might cause the float to slide upward along the tube. However, this W movement would be acceptable because the centrifugation valve 435 does not permit the movement of fluid through the buffy coat passage 487.

[0106] FIG. 4C is a cross-sectional view of another exemplary embodiment of a separator float similar to the apparatus of FIG. 4A. Here, the separator float 420 includes a main body portion 422 having a first end 424 and a second end 426. The main body portion has an outer diameter 438, while the sample tube 410 has an inner diameter 417. Again, there is no pressure relief passage 487. Rather than pressure seals 404, at least one support member extends radially outwards from the main body portion towards the sample tube 410, and has a diameter substantially equivalent to the inner diameter 417. Here, two support members 440 are located at the first end 424 and the second end 426. The support member(s) can also be described as being circumferentially disposed about the main body portion. The support members are centrifugation valves, as described above. The support member(s) and the main body portion 422 define an annular volume 470. The centrifugation valve(s) itself can be considered to have an annular shape when considered in isolation. The centrifugation valves are oriented to allow fluid to flow through the annular volume 470 from second end 426 to first end 424 during centrifugation. When centrifugation ends, the centrifugation valves close, no longer permitting fluid flow through the annular volume. It is again contemplated that any pressure difference between the first end 424 and the second end 426 would result only the float sliding upward along the tube.

[0107] FIG. 4D is a cross-sectional view of yet another exemplary embodiment of a separator float of the present disclosure. The separator float 420 is similar to the float of FIG. 4A, except the float 420 does not include a buffy coat passage 478 or a centrifugation valve 435. It is contemplated that the separator float initially begins near the open end 416 of the sample tube. During centrifugation, the float is pushed downwards into the blood. The resulting pressure on the first one-way pressure relief valve 434 is sufficient to open the valve, allowing blood components to flow from one end to the other. The various components then settle into layers based on density, with the target cells residing within the pressure relief passage 487. After centrifugation, the relief valve remains closed, keeping the target cells in the pressure relief passage 487.

[0108] Another concept is illustrated in FIGs. 5A-C. FIG. 5A is a side view showing a blood separation apparatus 500 including a sample tube 510 and a separator float 520. The sample tube 510 is formed from a sidewall 512 and has a first, closed end 514 and a second, open end 516.

[0109] FIG. 5B is a perspective view of the float in a closed position, and FIG. 5C is a view of the float in an open position. The separator float 520 is formed from a first piece 540 and a second piece 550. The float 520 has a first end 526 and a second end 524. The first piece 540 has a first end 546 and a second end 544. Similarly, the second piece 550 has a first end 556 and a second end 554. The pieces are joined together at their first end 546, 556, for example by a hinge 574. The first piece 540 and second piece 550 cooperate to form a rectangular passage 584 between the first end 526 and second end 524 of the float 520. The float 520 contains a slide 508 within the rectangular passage 584. FIG. 5B shows the float 520 after removal from the sample tube. FIG. 5C shows the float 520 after the float 520 has been opened and the slide has been removed.

[0110] In use, the slide 508 is placed in the float 520, and the float is centrifugated with the blood sample. Buffy coat constituents present in the rectangular passage 584 adhere to the slide 508 within the rectangular passage 584. After centrifugation, the float 520 may be removed from the sample tube 510 and opened at the hinge 574 to extract the slide 508. The slide, with the buffy coat constituents already adhered to it, can then be examined.

[0111] In other specific embodiments, it is contemplated that the float 520 can be used with the flexible bag 302, shown in FIG. 3. In these embodiments, the flexible bag 302 is placed in the rectangular passage 584 instead of the slide 508.

[0112] FIG. 6 illustrates still another concept. A side view of a blood separation apparatus 600 includes a sample tube 610 and a two-piece float 620. The sample tube 610 is formed from a sidewall 612 and has a first, closed end 614 and a second, open end 616.

[0113] The two-piece float 620 includes a top float 660 and a bottom float 668. The top float 660 includes a lower support member 662 having an upper surface 663 and a lower surface 664.

[0114] The top float 660 is formed from a lower support member 662 and a pitot tube 690. The lower support member 662 has an upper surface 663 and a lower surface 664. The pitot tube 690 extends from the lower surface 664 through the upper surface 663 and has a top end 694 located distally from the upper surface 663. The pitot tube forms a passage from the lower surface 663 to the top end 694. In this respect, a pitot tube acts like a straw; fluid flows through the pitot tube when the pressure at the top of the pitot tube is lower than the pressure at the bottom of the pitot tube.

[0115] The bottom float 668 may comprise a support member 670 having an upper surface 671 and a lower surface 672. The top float lower support member lower surface 664 and the bottom float support member upper surface 671 are complementarily shaped.

[0116] Generally, the density of the top float 660 and the bottom float 668 are independently from about 1 .029 to about 1 .09. The top float 660 has a density intermediate that of plasma and the buffy coat constituents, or in other words a specific gravity of from about 1 .029 to about 1.08. The bottom float 668 has a density intermediate that of the buffy coat constituents and red blood cells, or in other words a specific gravity of from about 1 .08 to about 1 .09. Regardless of the value of the density, it is generally contemplated in specific embodiments that the top float has a density which is less than the bottom float.

[0117] In use, the apparatus of FIG. 6 traps buffy coat constituents in the volume 685 between the lower surface 664 of the lower support member 662 of the top float 660 and the upper surface 671 of the support member 670 of the bottom float 668. The top float 660 is then pushed downwards towards the bottom float 668 to extract buffy coat constituents through the pitot tube 690.

[0118] In these embodiments, the pitot tube 690 is integral with the lower support member 662.

[0119] However, it is also contemplated that the pitot tube is made separately from the lower support member. In such embodiments, only the lower support member 662 of the top float 660 is centrifuged with the blood sample. The lower support member in this case is made with a first passage from the lower surface 664 to the upper surface 663. After centrifugation ends, the pitot tube 690 is inserted to engage the first passage. The top float 660 is then pushed down towards the bottom float 668 to push buffy coat constituents through the pitot tube 690.

[0120] Some additional variations on this two-piece float 620 are contemplated. The pitot tube 690 itself is contemplated as being rigid, so as to be suitable for use in pushing the top float 660 downwards. In some embodiments, however, and as depicted here, the top float 660 may further comprise a manipulator 666, such as a handle, extending axially from the upper surface 663 of the top float 660, and this manipulator can be used to push the top float downwards. The manipulator is generally made or situated on the upper surface 663 so that its presence will not affect the final alignment of the lower support member 662 with the buffy coat constituents.

[0121] After centrifugation, excess pressure may form below the bottom float 668. This pressure may be relieved through a pressure relief tube 676 which extends axially from the tower surface 672 of the bottom float support member 670 through the upper surface 671 and terminates at an upper end 677. The top float 660 includes a second passage 675 from the lower surface 664 to the upper surface 663, and the pressure relief tube 676 extends through the second passage.

[0122] In addition, if desired, an axial support member 673 may extend axially from the lower surface 672 of the support member 670 of the bottom float 668. It is contemplated that this axial support member 673 would contact the closed end 614 of the sample tube 610 and provide additional resistance when the top float 660 is pushed towards the bottom float 668. It should be noted, however, that the length of the axial support member may be uncertain, as the level at which the bottom float support member 670 rests after centrifugation would depend partially on the size of the blood sample, and the size of the blood sample with which the float is used is not a factor that can be controlled during manufacture of the float.

[0123] FIGS. 7A-7C show three exemplary embodiments of another concept. Here, a blood separation apparatus 700 includes a sample tube 710 and a two piece float 720. The sample tube 710 is formed from a sidewall 712 and has a first, closed end 714 and a second, open end 716. Generally speaking, the buffy coat is captured between the two pieces of the float.

[0124] The two-piece float can include a recess, in which the buffy coat layer is trapped or contained. The recess is placed in different locations in FIG. 7A and FIG. 7B.

[0125] A first exemplary embodiment is shown in FIG. 7A. The two-piece float 720 comprises a top float 760 and a bottom float 768. The top float 760 includes a lower lateral support member 762 which has an upper surface 763 and a lower surface 764. A member or manipulator 766 extends axially from the upper surface 763 of the lower lateral support member 762. The manipulator 766 is used to push the top float 760 towards the bottom float 768. [0126] The bottom float 768 comprises an upper lateral support member 770 having an upper surface 771 and a lower surface 772. The top float lower lateral support member 762 and the bottom float upper lateral support member 770 are complimentarily shaped to form a recess 796. For example, FIG. 7A shows five sides of the recess 796 being formed in the bottom float upper lateral support member 770, while the top float lower lateral support member 762 covers the recess. It is contemplated that this arrangement could be reversed, with the top float lower lateral support member 762 containing the recess and the bottom float upper lateral support member 770 covering the recess. The recess resides in a lateral plane, in other words perpendicularly to the long axis of the sample tube 710.

[0127] A second exemplary embodiment is shown in FIG. 7B. Here, the recess 796 is located in the member 766. The hollow member 766 is open on the lower surface 764 of the lower lateral support member 762, and is adapted to receive the buffy coat layer. The upper lateral support member 770 of the bottom float 768 is used to seal the hollow member.

[0128] In FIG. 7C, there is no recess. Rather, the buffy coat layers are simply trapped between the top float 760 and the bottom float 768.

[0129] Generally, the density of the top float 760 and the bottom float 768 are independently from about 1.029 to about 1.09. The top float 760 has a density intermediate that of plasma and the buffy coat constituents, or in other words a specific gravity of from about 1.029 to about 1.08. The bottom float 768 has a density intermediate that of the buffy coat constituents and red blood cells, or in other words a specific gravity of from about 1.08 to about 1.09. Regardless of the value of the density, it is generally contemplated in specific embodiments that the top float has a density which is less than the bottom float. Preferably, the densities are selected in FIG. 7A and FIG. 7B so that there is little to no additional space between the two floats, so that the buffy coat is located in the recess.

[0130] In use, the apparatus of FIGS. 7A-7C traps buffy coat constituents in the volume between the lower surface 764 of the lower support member 762 of the top float 760 and the upper surface 771 of the support member 770 of the bottom float 768. The buffy coat constituents are then removed from the float. In the case of FIG. 7A and FIG. 7B, this may be done by breaking the sample tube to retrieve the float. In the case of FIG. 7C, one end of the sample tube is broken off, and one piece of the two-piece float is then removed so that the buffy coat constituents can be drained out of the remainder of the tube.

[0131] When the apparatuses of FIGS. 7A and B is used to separate buffy coat constituents, after centrifugation, the buffy coat constituents reside in the volume between the lower surface 764 of the lower lateral support member 762 of the top float 760 and the upper surface 771 of the upper lateral support member 770 of the lower float 768. The member or manipulator 766 is pushed down to push buffy coat constituents in the recess 796. The float can then be separated from the sample tube, and the buffy coat can be extracted onto a slide for examination.

[0132] In particular embodiments, it is contemplated that a suitable slide 708 could be placed within the recess 796, and the buffy coat constituents could adhere to the slide 708 within the recess 796. The two-piece float 720 could then be separated from the sample tube 710 and the slide 708 would be removed from the recess 796 in the two-piece float 720. The slide, with the buffy coat constituents already adhered to it, could then be examined.

[0133] Some additional variations on this two-piece float 720 are contemplated. After centrifugation, excess pressure may form below the bottom float 768. This pressure may be relieved through a pressure relief tube 776 which extends axially from the lower surface 772 of the bottom float upper lateral support member 770 through the upper surface 771 and terminates at an upper end 777. The top float 760 includes a second passage 775 from the lower surface 764 to the upper surface 763, and the pressure relief tube 776 extends through the second passage.

[0134] In addition, if desired, an axial support member 773 may extend axially from the lower surface 772 of the upper lateral support member 770 of the bottom float 768. It is contemplated that this axial support member 773 would contact the closed end 714 of the sample tube 710 and provide additional resistance when the top float 760 is pushed towards the bottom float 768. It should be noted, however, that the length of the axial support member may be uncertain, as the level at which the bottom float support member 770 rests after centrifugation would depend partially on the size of the blood sample, and the size of the blood sample with which the float is used is not a factor that can be controlled during manufacture of the float

[0135] FIG. 8 and FIG. 9 illustrate another concept with two exemplary embodiments. Briefly, the separator is a two-piece float. The upper piece is used to W 201 push fluid down towards the lower piece. The lower piece contains a pitot tube through which the buffy coat constituents are extracted from the tube.

[0136] FIG. 8 shows a side view of a sample tube 810 and a separator float 820. The sample tube 810 is formed from a sidewall 812 and has a first, closed end 814 and a second, open end 816.

[0137] The separator float 820 includes a lower piece 862 and an upper piece 860. The lower piece 862 is formed from a main body portion 822. The main body portion 822 has a top end 824 and a bottom end 826. One or more support members 827 protrude from the main body portion. The main body portion and the support members define an annular volume 880. A pitot tube 890 extends axially from the top end 824 of the main body portion 822. The main body portion contains an internal passage 882 that connects a distal end 892 of the pitot tube 890 to an opening 833 in the annular volume 880. In particular embodiments, the main body portion has a specific gravity of from about 1.029 to about 1.09, including from about 1.08 to about 1.09.

[0138] The upper piece 860 includes a passageway 865 through which the pitot tube 890 extends. The upper piece also includes a member 866 or handle extending axially away from the main body portion 822. The upper piece 860 has a lower density than the main body portion 822, i.e. is less dense. The upper piece and the lower piece both have a diameter sufficient to engage the sidewall 812 of the sample tube 810. Put another way, the upper piece 860 and the lower piece 862 both have substantially the same diameter.

[0139] In some embodiments, the passageway 865 is located inside the member 866, i.e. the member is hollow or the member 866 surrounds the pitot tube 890. The float 820 may further include one or more one-way valves 834 in the opening 833 of the main body portion which are oriented to permit flow from the annular volume 880 into the internal passage 882. Put another way, the one-way valves are oriented to open when the pressure in the annular volume is greater than the pressure in the internal passage. In other embodiments, the internal passage 882 connects the pitot tube 890 to a plurality of openings in the annular volume 880.

[0140] When the apparatus of FIG. 8 is used, both the upper piece 860 and the lower piece 862 are aligned with each other so that the pitot tube 890 extends through the passageway 865, and both pieces are placed into the sample tube with the blood sample prior to centrifugation. During centrifugation, the main body portion 822 aligns with the buffy coat constituents and traps them in the annular volume 880 after centrifugation ends. The upper piece 860, which has a lower density than the main body portion, remains floating and not in contact with the main body portion. The upper piece 860 is then pushed downwards towards the main body portion to force fluid into the annular volume, which in turn pushes the buffy coat constituents upwards through the pitot tube 890. The one-way valves 834 can be used to keep fluid from entering the internal passage 882, particularly during centrifugation. In these embodiments, the pitot tube 890 is integral with the main body portion 822.

[0141] However, it is also contemplated that the pitot tube is made separately from the main body portion. In such embodiments, only the main body portion 822 is centrifuged with the blood sample. The internal passage 882 connects the opening 833 in the annular volume 880 to the top end 824 of the main body portion. After centrifugation ends, the pitot tube 890 is inserted to engage the internal passage 882 at the top end 824. The upper piece 860 is then threaded onto the pitot tube, so that the pitot tube extends through the member 866, and the upper piece 860 is pushed down towards the main body portion 822 to force fluid into the annular passage and push buffy coat constituents through the pitot tube 890. It should be noted that in addition, the upper piece 860 does not need to have a lower density than the lower piece 862 because the upper piece 860 is not centrifuged.

[0142] An optional pressure relief passage 887 may also be present for relieving excessive pressure. The pressure relief passage 887 is an independent passage between the top end 824 and the bottom end 826, and does not intersect the internal passage 882, any openings 833, or any one-way valves 834.

[0143] In specific embodiments, the one or more support members 827 includes a bottom support member 830 which protrudes from the bottom end 826 of the main body portion. The opening 833 may be located proximally to the bottom support member 830. The one or more support members 827 may also include a top support member 828 which protrudes from the top end 824 of the main body portion, i.e. which is proximal to the top end 824 of the main body portion.

[0144] FIG. 9 is a side view of a second exemplary embodiment similar to FIG. 8. Here, the one or more support members 827 include a bottom support member 830 which protrudes from the bottom end 826 of the main body portion. Here, the top support member 828 is a helical support member which is proximal to the top end 824 of the main body portion. One advantage to using a helical support member instead of a flat support member is that when the upper piece 860 is pushed towards a flat support member as in FIG. 8, fluid is forced into the annular volume 880 around the entire perimeter of the support member. This can cause mixing of the desired buffy coat constituents with the fluid, increasing the volume which must be extracted through the pitot tube and analyzed later for target cells. However, when the upper piece is pushed towards a helical support member, the fluid enters the annular volume along a smaller periphery and acts like a wave front that "pushes" the buffy coat into the pitot tube. The smaller periphery reduces the volume in which mixing occurs.

[0145] While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are or may be presently unforeseen may arise to applicants or other skilled in the art. Accordingly, the appended claims as filed and as they are amended are intended to embrace all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

CLAIMS:

1. A volume-occupying separator float, the float comprising:

a first piece and a second piece, each piece having a first end, a second end, an exterior surface, and an interior surface;

wherein the interior surfaces of the first and second pieces cooperate to form an open passage extending between the first end and the second end; and

wherein the first and second pieces can be connected together.

2. The float of claim 1 , wherein the first and second pieces have substantially the same three-dimensional shape.

3. The float of claim 1 , wherein the interior surface of the first piece comprises a semi-cylindrical surface.

4. The float of claim 1 , wherein a lateral cross-sectional view of the first piece has a semi-annular shape.

5. The float of claim 1 , wherein the interior surface of the first piece is substantially planar.

6. The float of claim 1 , wherein a lateral cross-sectional view of the interior surface of the first piece is substantially a straight line.

7. The float of claim 1 , wherein a lateral cross-sectional view of the interior surface of the first piece is substantially a straight line, and wherein a lateral cross-sectional view of the interior surface of the second piece is substantially a straight line with a central indent.

8. The float of claim 1 , wherein a lateral cross-sectional view of the open passage has a rectangular shape.

9. The float of claim 1 , wherein a lateral cross-sectional view of the open passage has a circular shape.

10. The float of claim 1 , wherein the exterior surface of the first and second pieces each substantially conform to an inner surface of the sidewalk

1 1. The float of claim 1 , wherein the exterior surface of the first and second pieces each comprise at least one support member for engaging an inner surface of the sidewall.

12. The float of claim 1 1 , wherein the exterior surface of the first and second pieces each have two support members.

13. The float of claim 1 , wherein the first piece and the second piece each have a specific gravity of from about 1.029 to about 1.09.

14. The float of claim 1 , wherein the first piece and the second piece each have a specific gravity of from about 1 .040 to about 1 .070.

15. The float of claim 1 , wherein the first piece and the second piece each have a specific gravity of from about 1 .08 to about 1.09.

16. The float of claim 1 , wherein the first piece and the second piece are each formed from a rigid plastic material.

1 7. The float of claim 1 , wherein the first and second pieces can be joined together using clips.

18. The float of claim 1 , wherein the first and second piece each further comprise at least one side surface.

19. The float of claim 18, wherein the first and second pieces can be connected together on the at least one side surface.

20. A method of capturing buffy coat constituents in a blood sample, comprising:

introducing a blood sample into a flexible bag;

placing a float around the flexible bag, the float having a first end and a second end and the float having a specific gravity intermediate that of plasma and red blood cells;

placing the flexible bag and float in a flexible sample tube, the sample tube having a sidewall, wherein the float engages the sidewall;

centrifuging the sample tube at a rotational speed that causes enlargement of the sidewall to a diameter sufficiently large to permit axial movement of the float, separation of the blood into discrete layers, and movement of the float into alignment with at least the buffy coat constituents;

reducing the rotational speed to cause the sidewall to capture the float; and

sealing the flexible bag at the first end and the second end of the float to capture the buffy coat constituents.

21. The method of claim 20, wherein the flexible bag is sealed by welding the first end and the second end of the float.

22. The method of claim 21 , wherein the welding is performed ultrasonically.

23. The method of claim 20, wherein the float comprises a first piece and a second piece, each piece having an exterior surface and an interior surface, the interior surfaces of the first and second pieces cooperating to form an open passage extending between the first end and the second end for the flexible bag.

24. The method of claim 23, wherein the first and second pieces have substantially the same three-dimensional shape.

25. The method of claim 23, wherein the interior surface of the first piece comprises a semi-cylindrical surface.

26. The method of claim 23, wherein a lateral cross-sectional view of the first piece has a semi-annular shape.

27. The method of claim 23, wherein the interior surface of the first piece is substantially planar.

28. The method of claim 23, wherein a lateral cross-sectional view of the interior surface of the first piece is substantially a straight line.

29. The method of claim 23, wherein the first and second pieces are separate.

30. The method of claim 23, wherein the first and second pieces are joined together on at least one side.

31. The method of claim 30, wherein the first and second pieces are joined together on two sides.

32. The method of claim 30, wherein the first and second pieces are joined together using clips.

33. The method of claim 30, wherein the first and second pieces are joined at the first end of the float.

34. The method of claim 23, wherein a lateral cross-sectional view of the interior surface of the first piece is substantially a straight line, and wherein a lateral cross-sectional view of the interior surface of the second piece is substantially a straight line with a central indent.

35. The method of claim 23, wherein a lateral cross-sectional view of the open passage has a rectangular shape.

36. The method of claim 23, wherein a lateral cross-sectional view of the open passage has a circular shape.

37. The method of claim 23, wherein the exterior surface of the first and second pieces each substantially conform to an inner surface of the sidewall.

38. The method of claim 23, wherein the exterior surface of the first and second pieces each comprise at least one support member for engaging an inner surface of the sidewall.

39. The method of claim 38, wherein the exterior surface of the first and second pieces each have two support members.

40. The method of claim 23, wherein the first and second pieces of the float each have a specific gravity of from about 1.029 to about 1.089.

41. The method of claim 23, wherein the first and second pieces of the float are each formed from a rigid plastic material.

42. The method of claim 20, further comprising combining the blood sample with a stain.

43. The method of claim 20, wherein the sample tube is seamless at least along the path of axial movement of the float.

44. The method of claim 20, wherein the flexible bag is formed of a transparent polymeric material.

45. A two-piece float for capturing buffy coat constituents of a blood sample, comprising:

a top float having a density intermediate that of plasma and the buffy coat constituents, wherein the top float comprises (i) a lower support member having an upper surface and a lower surface, and (ii) a pitot tube extending axially from the lower surface of the top float through the upper surface and having a top end located distally from the upper surface; and

a bottom float having a density intermediate that of the buffy coat constituents and red blood cells;

wherein the top float lower support member lower surface and the bottom float are complementarily shaped.

46. The two-piece float of claim 45, wherein the top float further comprises a second passage extending from the lower surface to the upper surface of the lower support member;

wherein the bottom float comprises (i) a support member having an upper surface and a lower surface, and (ii) a pressure relief tube extending axially from the lower surface of the bottom float support member through the upper surface and terminating at an upper end; and

wherein the upper end of the bottom float pressure relief tube extends through the top float second passage.

47. The two-piece float of claim 45, wherein the top float has a specific gravity of from about 1.029 to about 1.09.

48. The two-piece float of claim 45, wherein the bottom float has a specific gravity of from about 1.029 to about 1.09.

49. The two-piece float of claim 45, wherein the top float further comprises a manipulator extending axially from the upper surface of the lower support member.

50. The two-piece float of claim 45, wherein the top float and the bottom float are each formed from a rigid plastic material.

51. A method of capturing buffy coat constituents in a blood sample, comprising:

introducing the blood sample into a sample tube, the sample tube having a sidewall;

introducing a two-piece float into the sample tube, the two-piece float comprising:

wherein the top float lower support member lower surface and the bottom float are complementarily shaped;

wherein the top float lower support member and the bottom float engage the sidewall;

centrifuging the sample tube at a rotational speed that causes enlargement of the sidewall to a diameter sufficiently large to permit axial movement of the float, separation of the blood into discrete layers, and alignment of the buffy coat constituents between the top float and the bottom float; and

pushing the top float toward the bottom float to remove the buffy coat constituents through the pitot tube.

52. The method of claim 51 , wherein the top float further comprises a second passage extending from the lower surface to the upper surface of the lower support member;

53. The method of claim 51 , wherein the top float has a specific gravity of from about 1.029 to about 1.09.

54. The method of claim 51 , wherein the bottom float has a specific gravity of from about 1.029 to about 1.09.

55. The method of claim 51 , wherein the top float further comprises a manipulator extending axially from the upper surface of the lower support member, the manipulator being used to push the top float toward the bottom float.

56. The method of claim 51 , wherein the float is introduced into the sample tube before the blood sample is introduced therein.

57. The method of claim 51 , wherein the blood sample is introduced into the sample tube before the float is introduced therein.

58. The method of claim 51 , wherein the sample tube is sized to receive a blood sample of approximately ten milliliters in volume.

59. The method of claim 51 , wherein the blood sample comprises anticoagulated whole blood.

60. The method of claim 51 , wherein the sample tube is self-supporting.

61. The method of claim 51 , further comprising combining the blood sample with a stain.

62. The method of claim 51 , wherein the sample tube is seamless at least along the path of axial movement of the float.

63. The method of claim 5 , wherein the flexible sample tube is formed of a flexible polymeric material.

64. The method of claim 63, wherein the flexible sample tube is semi- transparent.

65. The method of claim 63, wherein the flexible sample tube is transparent.

66. A method of capturing buffy coat constituents in a blood sample, comprising:

a top float having a density intermediate that of plasma and the buffy coat constituents, wherein the top float comprises (i) a lower support member having an upper surface and a lower surface, and (ii) a first passage from the lower surface of the lower support member to the upper surface of the lower support member; and

engaging a pitot tube with the first passage of the top float;

67. A two-piece float for capturing buffy coat constituents of a blood sample, comprising:

a top float having a density intermediate that of plasma and the buffy coat constituents, wherein the top float comprises (i) a lower lateral support member having an upper surface and a lower surface, and (ii) a manipulator extending axially from the upper surface of the lower lateral support member; and a bottom float having a density intermediate that of the buffy coat constituents and red blood cells, wherein the bottom float comprises an upper lateral support member having an upper surface and a lower surface.

68. The float of claim 67, wherein the top float lower lateral support member and the bottom float upper lateral support member are complementarily shaped to form a recess.

69. The float of claim 68, further comprising a slide in the recess.

70. The float of claim 68, wherein the recess is substantially formed in the bottom float upper lateral support member, and the top float lower lateral support member covers the recess.

71. The float of claim 68, wherein the recess is substantially formed in the top float lower lateral support member, and the bottom float upper lateral support member covers the recess.

72. The float of claim 68, wherein the recess includes an opening on the lower surface of the lower lateral support member and extends axially from the upper surface of the lower lateral support member.

73. The float of claim 67, wherein the bottom float further comprises a support member extending axially from the lower surface of the upper lateral support member.

74. The float of claim 67, wherein the top float has a specific gravity of from about 1 .029 to about 1.09.

75. The float of claim 67, wherein the bottom float has a specific gravity of from about 1.029 to about 1.09.

76. The float of claim 67, wherein the top float and the bottom float are each formed from a rigid plastic material.

77. A method of capturing buffy coat constituents in a blood sample, comprising:

a top float having a density intermediate that of plasma and the buffy coat constituents, wherein the top float comprises (i) a lower lateral support member having an upper surface and a lower surface, and (ii) a manipulator extending axially from the upper surface of the lower lateral support member; and

a bottom float having a density intermediate that of the buffy coat constituents and red blood cells, wherein the bottom float comprises an upper lateral support member having an upper surface and a lower surface;

wherein the top float lower support member and the bottom float upper lateral support member engage the sidewall;

retrieving the buffy coat constituents.

78. The method of claim 77, wherein the bottom float further comprises a support member extending axially from the lower surface of the upper lateral support member.

79. The method of claim 77, wherein a recess is formed by the top float lower lateral support member and the bottom float upper lateral support member; and wherein the buffy coat constituents are retrieved by pushing the top float toward the bottom float to push the buffy coat constituents into the recess and separating the two piece float from the sample tube.

80. The method of claim 79, wherein the recess is substantially formed in the bottom float upper lateral support member and the top float lower lateral support member covers the recess.

81. The method of claim 79, wherein the recess is substantially formed in that the top float lower lateral support member and the bottom float upper lateral support member covers the recess.

82. The method of claim 79, wherein the recess includes an opening on the lower surface of the lower lateral support member and extends axially from the upper surface of the lower lateral support member.

83. The method of claim 79, wherein a slide is located in the recess.

84. The method of claim 77, wherein the top float has a specific gravity of from about 1.029 to about 1.09.

85. The method of claim 77, wherein the bottom float has a specific gravity of from about 1.029 to about 1.09.

86. The method of claim 77, wherein the float is introduced into the sample tube before the blood sample is introduced therein.

87. The method of claim 77, wherein the blood sample is introduced into the sample tube before the float is introduced therein.

88. The method of claim 77, wherein the sample tube is sized to receive a blood sample of approximately ten milliliters in volume.

89. The method of claim 77, wherein the blood sample comprises anticoagulated whole blood.

90. The method of claim 77, wherein the sample tube is self-supporting.

91. The method of claim 77, further comprising combining the blood sample with a stain.

92. The method of claim 77, wherein the sample tube is seamless at least along the path of axial movement of the float.

93. The method of claim 77, wherein the flexible sample tube is formed of a flexible polymeric material.

94. The method of claim 93, wherein the flexible sample tube is semi- transparent.

95. The method of claim 93, wherein the flexible sample tube is transparent.

96. A volume-occupying separator float, comprising:

a main body portion having a top end and a bottom end; one or more support members protruding laterally from the main body portion;

wherein the main body portion and the one or more support members define an annular volume;

a pitot tube extending axially from the top end of the main body portion;

an internal passage that passes through the main body portion and connects the pitot tube to an opening in the annular volume; and

an upper piece comprising (i) a passageway through which the pitot tube extends, and (ii) a member extending axially away from the main body portion;

wherein the upper piece has a lower density than the main body portion.

97. The float of claim 96, wherein the upper piece passageway is located inside the member.

98. The float of claim 96, further comprising a one-way valve in the opening oriented to permit flow from the annular volume into the internal passage.

99. The float of claim 96, wherein the main body portion internal passage connects the pitot tube to a plurality of openings in the annular volume.

100. The float of claim 96, wherein the main body portion further comprises a pressure relief passage extending from the first end to the second end, the pressure relief passage not intersecting the internal passage.

101. The float of claim 96, wherein one support member is located on the bottom end of the main body portion, and the opening connecting to the annular volume is located proximally to the one support member.

102. The float of claim 96, wherein the float comprises one support member extending laterally from a bottom end of the main body portion and at least one helical support member located proximal to the top end of the main body portion.

103. The float of claim 96, wherein the main body portion has a specific gravity of from about 1 .029 to about 1.09.

104. The float of claim 96, wherein the main body portion has a specific gravity of from about 1 .08 to about 1 .09.

105. The float of claim 96, wherein the float is formed from a rigid plastic material.

106. A method of capturing buffy coat constituents in a blood sample, comprising:

introducing the blood sample into a sample tube, the sample tube having an inner surface;

a lower piece comprising (i) a main body portion having a top end and a bottom end, (ii) one or more support members protruding laterally from the main body portion and engaging the inner surface of the sample tube, wherein the main body portion and the one or more support members define an annular volume; (iii) a pitot tube extending axially from the top end of the main body portion; and (iv) an internal passage that passes through the main body portion and connects the pitot tube to an opening in the annular volume; and

wherein the upper piece has a lower density than the main body portion; and

wherein the main body portion has a density intermediate that of red blood cells and plasma;

centrifuging the sample tube at a rotational speed that causes enlargement of the sidewall to a diameter sufficiently large to permit axial movement of the float, separation of the blood into discrete layers, and alignment of the annular volume with at least the buffy coat constituents; and

pushing the upper piece down to force fluid towards the annular volume and push the buffy coat constituents through the pitot tube.

107. The method of claim 106, wherein the upper piece passageway is located inside the member.

108. The method of claim 106, further comprising a one-way valve in the opening oriented to permit flow from the annular volume into the internal passage.

109. The method of claim 106, wherein the main body portion internal passage connects the pitot tube to a plurality of openings in the annular volume.

1 0. The method of claim 106, wherein the main body portion further comprises a pressure relief passage extending from the first end to the second end, the pressure relief passage not intersecting the internal passage.

1 1 1 . The method of claim 106, wherein one support member is located on the bottom end of the main body portion, and the opening connecting to the annular volume is located proximally to the one support member.

1 12. The method of claim 106, wherein the float comprises one support member extending laterally from a bottom end of the main body portion and at least one helical support member located proximal to the top end of the main body portion.

1 13. The method of claim 106, wherein the main body portion has a specific gravity of from about 1.029 to about 1.09.

1 14. The method of claim 106, wherein the main body portion has a specific gravity of from about 1.08 to about 1.09.

1 15. The method of claim 106, wherein the float is formed from a rigid plastic material.

1 16. The method of claim 106, wherein the float is introduced into the sample tube before the blood sample is introduced therein.

1 17. The method of claim 106, wherein the blood sample is introduced into the sample tube before the float is introduced therein.

1 18. The method of claim 106, wherein the sample tube is sized to receive a blood sample of approximately ten milliliters in volume.

1 19. The method of claim 106, wherein the blood sample comprises anticoagulated whole blood.

120. The method of claim 106, wherein the sample tube is self- supporting.

121 . The method of claim 106, further comprising combining the blood sample with a stain.

122. The method of claim 106, wherein the sample tube is seamless at least along the path of axial movement of the float.

123. The method of claim 106, wherein the flexible sample tube is formed of a flexible polymeric material.

124. The method of claim 123, wherein the flexible sample tube is semi- transparent.

125. The method of claim 123, wherein the flexible sample tube is transparent.

126, A method of capturing buffy coat constituents in a blood sample, comprising:

introducing a volume-occupying float into the sample tube, the float comprising:

a main body portion having a top end and a bottom end; one or more support members protruding laterally from the main body portion and engaging the inner surface of the sample tube;

wherein the main body portion and the one or more support members define an annular volume; and

an internal passage that passes through the main body portion and connects the top end of the main body portion to an opening in the annular volume;

centrifuging the sample tube at a rotational speed that causes enlargement of the sidewall to a diameter sufficiently large to permit axial movement of the float, separation of the blood into discrete layers, and alignment of the annular volume with at least the buffy coat constituents;

engaging a pitot tube with the internal passage at the top end of the main body portion;

threading an upper piece around the pitot tube, the upper piece engaging the inner surface of the sample tube, the upper piece comprising a hollow member that extends axially away from the main body portion and surrounds the pitot tube; and

127. The method of claim 126, further comprising a one-way valve in the opening oriented to permit flow from the annular volume into the internal passage.

128. The method of claim 126, wherein the main body portion internal passage connects the pitot tube to a plurality of openings in the annular volume.

129. The method of claim 126, wherein the main body portion further comprises a pressure relief passage extending from the first end to the second end, the pressure relief passage not intersecting the internal passage.

130. The method of claim 126, wherein one support member is located on the bottom end of the main body portion, and the opening connecting to the annular volume is located proximally to the one support member.

131 . The method of claim 126, wherein the float comprises one support member extending laterally from a bottom end of the main body portion and at least one helical support member located proximal to the top end of the main body portion.

132. A volume-occupying separator float, the float comprising;

a main body portion having a first end and a second end;

at least one pressure seal around the main body portion; and a buffy coat passage extending from the second end to the first end; and having a centrifugation valve at the second end, the centrifugation valve being oriented to open during centrifugation.

133. The float of claim 132, having a first pressure seal around the first end of the main body portion and a second pressure seal around the second end of the main body portion.

134. The float of claim 32, further comprising a pressure relief passage extending from the second end to the first end and having a one-way pressure relief valve oriented to open when pressure at the second end exceeds pressure at the first end by a first value.

135. The float of claim 132, wherein the float has a specific gravity of from about 1.029 to about 1.09.

136. The float of claim 132, wherein the float has a specific gravity of from about 1 .040 to about 1.070.

137. The float of claim 132, wherein the float has a specific gravity of from about 1 .08 to about 1.09.

138. The float of claim 132, wherein the float is formed from a rigid plastic material.

139. A method of capturing buffy coat constituents in a blood sample, comprising:

introducing the blood sample into a flexible sample tube, the sample tube having an inner surface;

introducing a volume-occupying float into the sample tube, said float having a specific gravity intermediate that of red blood cells and plasma;

said float comprising:

a main body portion having a first end and a second end; at least one pressure seal around the main body portion and engaging the inner surface; and

a buffy coat passage extending from the second end to the first end and having a centrifugation valve at the second end, the centrifugation valve being oriented to open during centrifugation;

centrifuging the sample tube at a rotational speed that causes enlargement of the sidewall to a diameter sufficiently large to permit axial movement of the float, separation of the blood into discrete layers, and capture of the buffy coat constituents in the buffy coat passage of the float, wherein the first pressure seal and the second pressure seal substantially prevent the blood sample from traveling between the float and the inner surface of the sample tube;

reducing the rotational speed to cause the inner surface to capture the float; and

removing the float from the sample tube.

140. The method of claim 39, wherein the float has a first pressure seal around the first end of the main body portion and a second pressure seal around the second end of the main body portion.

141. The method of claim 139, wherein the float further comprises a pressure relief passage extending from the second end to the first end and having a one-way pressure relief valve oriented to open when pressure at the second end exceeds pressure at the first end by a first value.

142. The method of claim 139, wherein the float has a specific gravity of from about 1.029 to about 1.09.

143. The method of claim 139_» wherein the float has a specific gravity of from about 1.040 to about 1.070.

144. The method of claim 139, wherein the float has a specific gravity of from about 1.08 to about 1.09.

145. The method of claim 139, wherein the float is formed from a rigid plastic material.

146. The method of claim 139, wherein the float is introduced into the sample tube before the blood sample is introduced therein.

147. The method of claim 139, wherein the blood sample is introduced into the sample tube before the float is introduced therein.

148. The method of claim 139, wherein the sample tube is sized to receive a blood sample of approximately ten milliliters in volume.

149. The method of claim 139, wherein the blood sample comprises anticoagulated whole blood.

150. The method of claim 139, wherein the sample tube is self- supporting.

151. The method of claim 139, further comprising combining the blood sample with a stain.

152. The method of claim 139, wherein the sample tube is seamless at least along the path of axial movement of the float.

153. The method of claim 139, wherein the flexible sample tube is formed of a flexible polymeric material.

154. The method of claim 153, wherein the flexible sample tube is semi- transparent.

155. The method of claim 153, wherein the flexible sample tube is transparent.

156. A volume-occupying separator float, the float comprising:

a main body portion having a top end and a bottom end; and at least one centrifugation valve circumferentially disposed about the main body portion;

wherein the centrifugation valve is configured to open during centrifugation.

157. The float of claim 156, wherein the at least one centrifugation valve is located at the top end of the main body portion.

158. The float of claim 156, wherein the at least one centrifugation valve is located at the bottom end of the main body portion. 59. The float of claim 156, having two centrifugation valves, wherein one centrifugation valve is located at the top end of the main body portion, and the other centrifugation valve is located at the bottom end of the main body portion.

160. The float of claim 156, wherein the float has a specific gravity of from about 1.029 to about 1.09.

161. The float of claim 156, wherein the main body portion is formed from a rigid plastic material.

162. A method of capturing buffy coat constituents in a blood sample, comprising:

said float comprising:

a main body portion having a top end and a bottom end; and a centrifugation valve circumferentially disposed about the main body portion, wherein the centrifugation valve is configured to open during centrifugation;

centrifuging the sample tube at a rotational speed that causes enlargement of the sidewall to a diameter sufficiently large to permit axial movement of the float, separation of the blood into discrete layers and to open the centrifugation valve, and capture of the buffy coat constituents in the buffy coat passage of the float;

reducing the rotational speed to cause the inner surface to capture the float, wherein the centrifugation valve closes when the rotational speed is reduced; and

removing the float from the sample tube.

163. The method of claim 162, wherein the at least one centrifugation valve is located at the top end of the main body portion.

164. The method of claim 162, wherein the at least one centrifugation valve is located at the bottom end of the main body portion.

165. The method of claim 162, having two centrifugation valves, wherein one centrifugation valve is located at the top end of the main body portion, and the other centrifugation valve is located at the bottom end of the main body portion.

166. The method of claim 162, wherein the float has a specific gravity of from about 1.029 to about 1.09.

167. The method of claim 162, wherein the float is formed from a rigid plastic material.

168. The method of claim 162, wherein the float is introduced into the sample tube before the blood sample is introduced therein.

169. The method of claim 162, wherein the blood sample is introduced into the sample tube before the float is introduced therein.

170. The method of claim 162, wherein the sample tube is sized to receive a blood sample of approximately ten milliliters in volume.

171. The method of claim 162, wherein the blood sample comprises anticoagulated whole blood.

172. The method of claim 162, wherein the sample tube is self- supporting.

173. The method of claim 162, further comprising combining the blood sample with a stain.

174. The method of claim 162, wherein the sample tube is seamless at least along the path of axial movement of the float.

175. The method of claim 162, wherein the flexible sample tube is formed of a flexible polymeric material.

176. The method of claim 175, wherein the flexible sample tube is semi- transparent.

177. The method of claim 175, wherein the flexible sample tube is transparent.

178. A volume-occupying separator float, the float comprising:

a main body portion having a first end and a second end;

at least one pressure seal around the main body portion; and a pressure relief passage extending from the second end to the first end and having a one-way pressure relief valve oriented to open when pressure at the second end exceeds pressure at the first end by a first value.

179. The float of claim 178, having a first pressure seal around the first end of the main body portion and a second pressure seal around the second end of the main body portion.

180. The float of claim 178, wherein the float has a specific gravity of from about 1.029 to about 1.09.

181 . The float of claim 178, wherein the float has a specific gravity of from about 1 .040 to about 1.070.

182. The float of claim 178, wherein the float has a specific gravity of from about 1.08 to about 1.09.

183. The float of claim 178, wherein the float is formed from a rigid plastic material.

184. A method of capturing buffy coat constituents in a blood sample, comprising:

said float comprising:

a pressure relief passage extending from the second end to the first end and having a one-way pressure relief valve oriented to open when pressure at the second end exceeds pressure at the first end by a first value;

removing the float from the sample tube.

185. The method of claim 184, wherein the float has a first pressure seal around the first end of the main body portion and a second pressure seal around the second end of the main body portion.

186. The method of claim 184, wherein the float has a specific gravity of from about 1.029 to about 1.09.

187. The method of claim 184, wherein the float has a specific gravity of from about 1.040 to about 1.070.

188. The method of claim 184, wherein the float has a specific gravity of from about 1.08 to about 1.09.

189. The method of claim 184, wherein the float is formed from a rigid plastic material.

190. The method of claim 184, wherein the float is introduced into the sample tube before the blood sample is introduced therein.

191. The method of claim 184, wherein the blood sample is introduced into the sample tube before the float is introduced therein.

192. The method of claim 184, wherein the sample tube is sized to receive a blood sample of approximately ten milliliters in volume.

193. The method of claim 184, wherein the blood sample comprises anticoagulated whole blood.

194. The method of claim 184, wherein the sample tube is self- supporting.

195. The method of claim 184, further comprising combining the blood sample with a stain.

196. The method of claim 184, wherein the sample tube is seamless at least along the path of axial movement of the float.

197. The method of claim 184, wherein the flexible sample tube is formed of a flexible polymeric material.

198. The method of claim 197, wherein the flexible sample tube is semi- transparent.

199. The method of claim 197, wherein the flexible sample tube is transparent.