DETAILED DESCRIPTION OF THE INVENTION

[0049] A first embodiment of the instant invention is illustrated in FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G. In accordance with the instant invention, a float 1 is used to form a step gradient within a vessel 2, as depicted in FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G. However, it should be understood that the float 1 also may be used to create a continuous gradient (not shown) where a density gradient is introduced gradually, and continuously changes along the height of the vessel. In FIG. 1A, the float 1 in the vessel 2 rises and floats on top of a liquid 3 introduced from a source 4. The diameter of the float 1 in the first embodiment preferably is slightly smaller than the inside diameter of the vessel 2.

[0050] The introduced liquid 3 contacts and then flows around the float 1, passes down between the outer surface of the float 1 and the inner surface of the vessel 2 and, as shown in FIG. 1A, produces a first zone 5. Typically, the zone 5 has the highest density of all the layers or zones, as is described in greater detail below. As shown in FIG. 1B, a second liquid 6 is introduced in a similar manner to produce zone 7. As shown in FIGS. 1C, 1D, and 1E, the procedure is repeated with succeeding less dense liquids 8, 10 and 12, to produce zones 9, 11 and 13.

[0051] A sample 14 to be analyzed within the vessel 2 is introduced last from a pipette 15 to produce zone 16 as shown in FIGS. 1F and 1G. Finally, as shown in FIG. 1G, the float 1 is removed by grasping a projecting pin 17 and lifting. Projecting pin 17 also can be manipulated following introduction of a liquid should a float require reorientation or repositioning in the vessel. The manipulation may be carried out between the addition of different liquids to prevent adhering or jamming of the float in the vessel. The vessel 2 depicted in FIG. 1G with sample and gradient may then be subjected to treatment by, for instance, insertion into a swinging bucket rotor of a centrifuge device. When a continuous gradient is required, the step gradient is prepared beforehand, and diffusion for a determined period of time used to convert the step gradient into a continuous one.

[0052] FIGS. 2A, 2B and 2C illustrate details of the float 1 and basic principles of operation of the float within the vessel 2. The float 1 and vessel 2 depicted in FIG. 2A are shown enlarged in FIG. 2B. The float 1 has an outer peripheral surface 20 that is shaped to conform to an inner surface of the vessel 2. For instance, the vessel 2 shown in the drawings is a tube having a circular cross-section when viewed from above or below. The float 1 has a corresponding circular shape with the outer peripheral surface 20 having a diameter that is smaller than the inner diameter of the surface of the vessel 2. Therefore, a gap G having a predetermined width is defined between the outer peripheral surface 20 of the float 1 and the inner surface of the vessel 2. The gap G may vary in size depending on the solutions to be introduced into the vessel 2 and the relative sizes of the float 1 and vessel 2.

[0053] In the depicted embodiment, the gap G is relatively small such that surface tension of the solution produces a capillary action within the gap G to maintain liquid in the gap and to prevent air bubbles in the gap. In many applications of the instant invention, the viscosity of fluid in the gap is sufficient to eliminate the possibility of turbulent fluid flow within the vessel 2 as the solution exits the gap and moves around the float 1. Therefore, mixing of layers of solution under the gap is almost non-existent and very sharp boundaries are produced between the zones, even with very small density increments.

[0054] It should be understood that a diminished rate of fluid flow is a desirable result of the instant invention depicted in FIGS. 1A-1G and FIGS. 2A-2C. The actual size of the gap G may be varied according to the viscosity of the liquids used and the size of the vessel 2. However, although capillary action and restricted rate of fluid flow are important to the instant invention, it is possible to use the float 1 of the instant invention without capillary action. For instance, the shape of the surfaces of the float 1 may be formed to discourage any increases in velocity of fluid moving over the surfaces of the float 1 to avoid turbulent flow of the fluids entering the vessel 2. The shape and surface contours of the float 1 are such that the flow of solution around the float 1 as the solution moves downward into the vessel 2 is minimal. Specifically, a upper surface 22 of the float 1 is tapered having a conical shape such that as fluid contacts the upper surface 22 viscous flow slows fluid motion as the fluid approaches an edge 23 of the float 1.

[0055] It should be understood that the upper surface 22 may have a more rounded shape when viewed from the side and need not be conical in shape so long as sufficient surface area is provided to allow the adhesive forces of the fluid to make contact with the upper surface 22 to moderate movement of the fluid. Edge 23 may also have a more rounded shape to facilitate movement of the fluid into the gap G so long as the flow rate comports with the intent of the instant invention. By rounding the edges, any adhering or jamming of the float in the vessel is minimized. The free floating nature is maintained. The outer peripheral surface 20 is parallel to a potion of the inner surface of vessel 2. Surface 20 preferably is long enough to prevent the float from tipping or spinning in the vessel, which movement could cause agitation of the fluids and disruption of the gradients.

[0056] It should also be understood that the vessel 2 and float 1 may have any of a variety of shapes when viewed in cross-section. The depicted vessel 2 is a tube having a circular cross-section. The vessel 2 also may have a square or triangular cross-sectional shape and the float 1 a corresponding square or triangular cross-sectional shape.

[0057] As shown in FIG. 2B, when a droplet 21 of solution is dropped from above the float 1, the droplet 21 is distributed circumferentially on upper tapered surface 22 and moves toward the edge 23, where the solution flows evenly into the gap G, and thereafter slowly moves on to the upper surface of the underlying layer of liquid. Velocity or speed of flow of the solution also is decelerated further as it flows around lower taper 25. Capillary forces and solution viscosity are sufficient to keep the velocity of the solution in the gap G to a minimum and further, regardless of the density of the liquids used, the gap G typically remains filled with solution due to the capillary action.

[0058] The float 1 is also formed with an upper integral pin 17 that allows the float 1 to be manipulated in the vessel as needed, and inserted and removed easily from the vessel 2. The density of the float 1 itself may be dictated by the choice of construction material or, as shown in FIG. 2C, an alternate embodiment of a float 1 a may be formed with a cavity 26 sealed by plug 27 to adjustably control the density of the float 1. Plug 27 may be constructed of a more dense material, such as a metal, to serve as a ballast to assist in maintaining the float in an upright orientation. The density is adjusted so as to be less than the least dense liquid to be added. Also, the cavity and hence, plug 27 can be altered in size to obtain a desired density and weight to the float. The floats 1 and 1 a can be constructed of an inert plastic material, such as polypropylene which has a density of approximately 0.95 g/cc, and the pin 17, being a small fraction of the mass of the float, may be either integrally molded into the float and of the same material, or may be another material such as polycarbonate or other plastic, and be inserted in a hole in the float as shown in FIGS. 4A and 4B.

[0059] Alternatively, the float can be made of a hydrophilic material. A hydrophilic surface might minimize any beading of liquid on the upper surfaces of the float. Thus, the float can be made of various organic materials, such as wood, hydrophilic plastic, rubber etc. or various inorganic materials, such as glass, ceramic, metal etc. Many of those substances are hydrophilic. The floats also may be made hollow or configured as shells filled with various filler materials of varying density to provide the artisan with additional means to vary the final weight and density of a float. Also, the float can contain a particular coating different from that of the material comprising the bulk of the float for a desired purpose. Thus, for example, a polypropylene float can have a hydrophilic plastic or a painted coating. Also, a surface can be treated to form a hydrophilic surface, such as, by plasma treatment, corona discharge or chromated sulfuric acid treatment. Also, a peripheral surface can be treated to be hydrophilic or less hydrophobic to prevent air from being trapped between the float and the inner surface of the vessel.

[0060] Further, the density of the float may be adjusted by inserting pins 17 having a variety of weights. For instance, a plurality of pins 17 may be produced, each pin 17 having a different mass for selectively adjusting the overall weight of the float. To enhance the density or weight of a float, the float may be constructed of two or more materials. Generally, a material with greater weight is situated in the bottom portion of the float to ensure proper orientation and buoyancy. Differential density or weight between the top and bottom portions of the float can be achieved by other means, such as inserting a weight in the bottom portion of the float, removing material from the upper portion of the float, such as having an air space below pin 17, or making the float out of two or more portions of different density, generally with the more dense materials located at the bottom portions of the float.

[0061] The shape of the various surfaces of the float 1 is not limited to the depictions in FIGS. lA-lG and FIGS. 2A-2C. FIGS. 3A, 3B and 3C illustrate alternative float designs. In FIG. 3A, a float 28 has upper edges 29 and lower edges 30 rounded to further assist in slowly accelerating flow at the upper edge, and decelerating flow at the lower edge as liquid flows over the underlying liquid. In FIG. 3B, float 31 has different upper edge 32 and lower edge 33, with the upper edge 32 sharp to help prevent air bubbles between the float and tube 2, and the lower edge 33 well rounded. Alternatively, upper edge 32 of FIG. 3B can be more rounded. In FIG. 3C, float 34 has a tip 35 extended to further control flow around the float. The shape of the lower surface of the float 34 and the tip 35 assist in keeping the interface 36 between two steps in the gradient sharp. Also, in FIG. 3C, tip 35 can be made of a material of greater density than that of the remainder of the float.

[0062] The inventors have tested and designed floats for Beckman Ultraclear tubes for the Beckman SW41 Ti rotor and for polycarbonate tubes for the Beckman SW28 rotor. For the SW 41 tubes, the floats were constructed of solid polypropylene, 13.1 mm in diameter with top and bottom tapers of 15 degrees, and were 6.35 mm high measured at the edge. Wall clearance was 0.25 mm (gap G). For the SW 28 rotor tubes, long and short versions of the floats were constructed which were 10.5 mm and 6 mm high at the edge, had clearances of 1 mm and 0.6 mm, with 15 degree tapers at the top and bottom. Holes through the float had 1.0-1.6 mm internal diameters, and the pins were made of 0.9 mm outside diameter polycarbonate monofilament. After the pins were inserted, one end was melted in a reducing flame to produce a ball at the tip, while the other end was heated to produce a small enlargement, which, when put into the float, sealed the pin in place.

[0063] All radial clearances kept the gap G between the float and the centrifuge tube wall (vessel 2) full of liquid at all densities used. Occasionally when floats were dropped into dry tubes, they became stuck at the bottom, Hence, the “round” at the bottom of the centrifuge tube is preferably filled with a “cushion”, i.e., the most dense gradient solution used, initially.

[0064] Experimentally, it was found that if the first 4-5 drops (circa 0.1 ml) of the liquid solution being added were introduced slowly over a period of 5-10 seconds, extraordinarily sharp interfaces were produced below the float. The remainder of the liquid gradient step then could be introduced more rapidly. Sharp interfaces were produced with the density difference between two steps being as little as 0.0017 g/ml.

[0065] The use of floats allows gradients to be formed as a series of short, well-defined zones that may be arranged to be linear, sigmoidal or of other gradient shape. If required, the gradients then can be evened by diffusion. The float/vessel arrangement allows the production of gradients that are more reproducible than those produced by conventional gradient makers, and allows many gradients to be made in parallel without requiring a multiplicity of gradient makers.

[0066] However, it also should be understood that by using the float of the instant invention, it is possible to quickly pour an amount of a fluid directly onto the top of the float and the fluid will gradually seep down around the float to create a layer fluid without significantly disturbing the layer or layers of fluid already beneath the float. Without the float, pouring of fluid into the vessel with previously introduced fluid layers in the vessel would guarantee mixing of the layers thereby making gradient layer formation impossible. Therefore, one important result possible by using any of the above described embodiments of the instant invention is that a density gradient can be produced quickly and reproducibly without concern of the rate of flow of any one liquid onto the upper surface of the float.

[0067] In yet another embodiment of the instant invention depicted in FIG. 5, a float 50 is positioned in a vessel 52 with the vessel 52 having an inner diameter that is significantly greater than the outer diameter of the float 50. The float 50 is similar to the float described above in FIGS. 1A-1G, but has a tube 55 attached to an upper surface of the float 50. The tube 55 is hollow and includes several apertures 58 for allowing the flow of fluid from within the tube 55 to an upper surface of the float 50. As fluid is introduced from the tube 55 via the apertures 58, the fluid contacts the upper surface of the float 50 and flows along the upper surface due to adhesion thereby slowly entering the vessel. Adhesion between the surfaces of the float 50 and the fluid slows velocity of the fluid such that the fluid forms a well-defined layer above previously introduced layers.

[0068] The upper surface 51 of the float 50 is preferably formed with only a slight incline to further inhibit acceleration of the fluid. The tube 55 attached to the fluid also may be used to raise and lower the float 50 with respect to the vessel 52. Specifically, a mechanical arm may be attached to the tube 55 to remotely control movement of the float in and out of the vessel 51. It should be understood that the tube 55 is flexible to allow movement of the float 50 upward as the vessel 52 is filled with fluid.

[0069] A fluid flow controller (not shown) preferably is used with the embodiment of the float 50 to control the amount of fluid introduced for each desired layer.

[0070] It should be understood that gradients may be used in many applications outside of the field of molecular biology. For instance, a vessel having a plurality of layers solution, each layer having a different density due to a specific concentration of solute in each layer, may be used determine the density of an unknown material. A sample of material of unknown density dropped into the vessel will settle in the layer having a like density thereby providing a means for determining density of the unknown material. For example, different classes of plastics have different densities. Small pieces of plastic may be tested easily by dropping one small sample into a vessel having a plurality of solutions, each solution having a predetermined density such that the plurality of layers define a stepped density gradient. The plastic particle will drop to a layer having the same density and will float above those layers having a greater density. Similarly, identification of a gemstone based on density can be conducted.

[0071] Hence, it can be seen that the vessel and the float can be of any size necessary to enable separation of the desired material or materials. Also, a float of the instant invention can be used merely to prepare a gradient for other purposes, such as esthetic purposes. Hence, liquids of differing density, and having one or more other differing characteristics can be used with a float of the instant invention to produce continuous or discontinuous gradients. Examples include decorations and liquid drinks.

[0072] The materials and methods of the instant invention can be used in the separation of cellular elements from samples of whole blood, blood products or diluted blood. For example, white blood cells can be obtained from blood by density gradient centrifugation. Suitable materials to effect separation of the cellular elements and particularly the nucleated cellular elements from blood include media that comprise colloidal silica, silica gel, sugars, such as sucrose, ficoll, and particular products such as Ficoll-Hypaque, Isopaque, LymphoPrep and Percoll. See, for example, Parish et al., Eur J Imm (1974) 4:808.

[0073] Generally single step gradients are produced by gently layering the blood cell suspension onto a high density medium. The preparation then is centrifuged at low speed to effect separation of the cells.

[0074] Alternatively, the blood cell suspension can be layered onto a linear gradient, for example, of bovine serum albumin prior to centrifugation. The blood cell suspension can be layered onto a discontinuous gradient, for example, of bovine serum albumin. The densities of the layers can be configured so that the various elements band at the interfaces of the layers.

[0075] To ensure that discrete, sharp layers and hence, tight banding of cells occurs, it is beneficial to ensure a sharp interface between the cell suspension, for example, blood, and the separation medium. That goal can be achieved with use of an apparatus of interest. A suitably sized float of interest is used. The float of interest rests atop the separation medium. The float of interest allows passage of the, for example, blood along the lateral sides thereof and along the inner surface of the centrifuge tube containing the medium, float and cell suspension with minimal turbulence to ensure formation of a discrete linear interface of cell suspension and separation medium.

[0076] All references cited herein are incorporated by reference in entirety.

[0077] Although the instant invention has been described with reference to the preferred embodiments, the invention is not limited to the details thereof. Various substitutions and modifications will occur to those of ordinary skill in the art and all such substitutions and modifications are intended to fall within the scope of the invention as defined in the appended claims.

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