BACKGROUND OF THE INVENTION

[0001] This invention relates generally to fluidic processing of biological samples for diagnostic purposes, sedimentation or centrifugal pelleting of suspended particulate matter, such as cells, separating particulate matter based on density, and enumerating particulates or cells by measurement of packed volume. More specifically, this invention relates to male fertility testing, and, in particular, sperm cell counting.

[0002] Worldwide, 10-20% of couples that attempt to conceive a new child have sub-optimal fertility. Difficulty in conceiving may be due to defects in either the male or the female reproduction system or a combination of the two, or due to other contributing factors. In approximately 40% of cases of infertility, the male partner is a contributing factor. The primary metrics available to evaluate male fertility are sperm count and motility. Sperm count is a concentration of sperm cells in semen and motility is a percentage of sperm cells capable of movement.

[0003] Conventional methods of evaluating male fertility comprise conducting clinical tests including microscopic examination to measure sperm count and motility. Semen samples for the clinical tests must be provided at the site of examination leading to privacy concerns for male subjects. Furthermore, providing a semen sample at the site of examination or in a clinical setting is widely perceived as awkward or embarrassing. This perception can deter male fertility testing for couples with difficulty conceiving despite the high prevalence of male fertility issues. A semen analysis test suitable for use in the home may be useful in cases where aversion to clinical conditions would otherwise deter testing. A few semen analysis test kits have been developed for use in the home, such as those in which a colored line is displayed when the concentration of sperm cells in a sample exceeds a particular number (e.g., 20 million per mL) or a color change is displayed when concentration of viable sperm cells in a sample exceeds a particular number (e.g., 10 million per mL). In these examples of test kits, the semen analysis tests provide a non-quantitative evaluation of sperm count. In cases where a low sperm count is correctable or sperm count varies over time, it may be desirable to have a quantitative estimate of the absolute sperm count and motility. SUMMARY OF THE INVENTION

[0004] The disclosed devices and methods are for estimation or quantitative measurement of particulate content in a biological sample, including estimation of cell, such as sperm cell, count or concentration by centrifugal sedimentation of cells in fluid, such as seminal fluid. The assay is performed using an enclosed sedimentation column of defined cross-sectional area and by measuring height of a pellet of compacted cells ("pellet") within the sedimentation column with aid of a metering marks or a scale bar along the sedimentation column. In one embodiment, the device includes a cartridge containing the sedimentation column as well as chambers for holding directing and metering fluid and for sedimenting particulates or cells. The sedimentation column comprises fluid of defined density to separate cell populations by density. The sedimentation column may also include portions of variable cross-sectional area allowing for a visual estimation or quantitative reading of the height of sedimented cells over a wider range of cell concentrations than possible with a fixed cross-sectional area. The device also includes or may be used with an instrument for centrifugally rotating a cartridge. Embodiments include multiple walls between chambers and valves in the walls to limit evaporation of a density medium and meter fluid.

[0005] Embodiments of the device can be used at home as home use test kits to estimate or measure sperm cell concentration and motile sperm cell concentration, aiding in diagnosis and monitoring of male fertility disorders and allowing users to avoid having to provide samples in a clinical setting. When used in a fertility context, the device and method allow for a quantitative evaluation of sperm count and motility. The user can get a more accurate estimate of the actual sperm count rather than just determining whether the sperm count is above or below a certain threshold. The user can, for example, determine if sperm count and motility is only somewhat low, and so may be more readily correctable. Similarly, the user can determine if the sperm counts vary over time, possibly allowing the user to identify causative factors for sub-optimal sperm count, and otherwise track times when sperm counts are higher. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is shows a schematic top view of a cartridge, showing chambers, walls, valves and two arms.

Fig. 2 illustrates schematically movement of sample fluid from a sample cavity to a

sedimentation column during use.

Fig. 3 is a side view cross-section and top view of a cartridge, showing chambers, walls, a hub, two arms, a cartridge bottom, a cartridge top and metering marks.

Fig. 4 illustrates schematically an initial state and a final state of sedimentation, through a density medium, of a pellet from fluid.

Fig. 5 illustrates a side cross-section view of an assembled cartridge including density medium and packaging options.

Fig. 6 is a side view cross-section and top view of a cartridge, containing fluid, and showing a first arm and a counterweighted second arm.

Fig. 7 is a representation of sedimentation of sperm before and after centrifugal rotation.

Fig. 8 is a flowchart of a method of use of a cartridge to perform an assay of fluid.

Fig. 9 illustrates a top view and a side view of a sedimentation column with a pellet, density medium and metering marks.

Fig. 10 illustrates a top view and a side view of an alternative embodiment of a sedimentation column comprising a lens.

Fig. 11 illustrates a top view and side cross-section view of an alternative embodiment of a sedimentation column comprising a taper.

Fig. 12 illustrates a top view and side cross-section view of an alternative embodiment of a sedimentation column comprising a density gradient.

Fig. 13 illustrates a side view of a cartridge hub and spin shaft, with mating options.

Fig. 14 illustrates a side view and bottom view of an alternative embodiment of a cartridge hub and spin shaft.

Fig. 15 illustrates a sedimentation column with a density medium and pellet. DETAILED DESCRIPTION OF THE INVENTION

[0006] All Figures are for specific embodiments, non-limiting of claimed scope.

[0007] Discussion herein describes exemplary scenarios, certain embodiments, and selected applications, all non-limiting of claimed scope.

[0008] Various embodiments of estimation or quantitative measurement or assay of sperm count, density and motility based on volume occupied by sperm cells packed into a column of defined cross-section following centrifugation are disclosed. Visually reading a packed volume or height of a sedimentation pellet provides an easy -to-read method for estimating or quantitative measurement of cell concentration, which may be applied to sperm cells.

[0009] Prior art methods and devices for measuring sedimentation pellets after centrifuge are not suitable for direct application by users without medical training. Methods for measuring blood makeup, such as red blood cell count are not suitable for use measuring sperm

concentration. For humans, the average concentration of red blood cells in blood is

approximately 100 times higher than the average sperm concentration in semen. Also, the considerably higher viscosity of semen prevents uptake of a defined volume of sample by capillary action as is necessary for operation of hematocrit tubes and retards or prevents sedimentation of sperm cells upon centrifugation. Semen is also highly heterogeneous in composition (i.e., initially contains regions of high and low sperm concentration), unlike blood, and therefore requires homogenization to achieve reproducible measurements of concentration. For these reasons, different fluidic structures and modified sample processing steps are necessary to form a sedimented pellet of sperm cells that can be measured. Furthermore, the prior art of hematocrit and blood analysis techniques requires heavy and expensive centrifuges or dedicated analyzers to spin and contain the sedimentation capillaries, making them impractical for the home use or the general public. Described embodiments of devices, methods and kits overcome the limitation of prior art.

[0010] In one embodiment, the estimation or quantitative assay of cell count is provided for through use of a device that is included in a kit. An embodiment device comprises a cartridge including a sedimentation column and a motorized instrument for spinning the cartridge. The cartridge may attach to a motorized spin shaft by using a frictional press or magnets. A claimed kit may comprise a fluid transfer and measuring device and a sample collection cup to assist in both pretreating the sample with enzyme and transferring the sample to a cartridge. The cartridge may be a disposable cartridge. Embodiments include a control fluid and a cartridge for use with this control.

[0011] Throughout this disclosure, exemplary scenarios include devices and methods for semen samples for fertility analysis. Other fluids and assays may be performed using the claimed devices, methods and kits. For example, the device and methods may be applied to motor oil assay or to automated quantification of red blood cells or leukocytes in blood. In some embodiments, the samples are food, soil or other materials. In yet other embodiments, the samples are biological samples, such as blood, stool, semen, and other samples that might come from an organism, such as a human. Various biological samples comprise cells that span a range of densities (e.g., semen includes sperm and leukocytes in many cases, or may include foreign microbes in the case of an infection; additionally, sperm cells themselves may vary in density) and a variety of solid or semi-solid particles (e.g., semen may include cellular fragments or gelled proteins). Density medium formulations in the prior art produce indistinct pellets upon centrifugation as a result of this heterogeneity. Prior art formulations fail repeatability and quantitative measurement requirements. Prior art formulations also fail practical and consistent readings due to decompaction of pellets after centrifugation. Prior art devices and methods may expose users, particularly non-medically trained users to biological, chemical or mechanical hazards, such diseases carried as bodily fluids, chemicals used in assay methods, and spinning mechanisms. Prior art devices and methods have limited shelf life. Prior art methods fail to limit evaporation of density medium.

[0012] The use of high osmolality density agents, such as cesium chloride, in combination with a saline buffer, may produce a density medium with an osmolarity that is 1.1 to 3 times more than mammalian body fluids such as human blood. A hyper-osmotic density medium shrinks cells and produces dense pellets when centrifuged. This effect increases the density difference between cells and low-density non-cellular particles, and may help to make the variable density of the cells more uniform. This also yields a pellet with a distinct edge that provides reliable and accurate reading by a user. Use of semen with prior art density media produces a pellet with an indistinct boundary.

[0013] A claimed density medium is formulated to create a hyperosmotic medium with a low enough index of refraction to maintain pellet visibility. [0014] Following centrifugation, pellets may spread out in prior art devices and methods, resulting in a pellet that produces variable results as it expands over time. For use in applications targeted by claimed devices and methods such prior art pellet expansion results in an unstable and thus unreliable and non-quantitative reading of the assay. The use of a long-chain surfactant in a media formulation, such as Pluronic® surfactants (BASF Corporation, 100 Park Ave, Florham Park, NJ 07932), maintains pellet compaction by increasing the adhesion between cells in the pellet, and allows a longer period of valid reading times. The addition of a surfactant in the density medium also enhances its behavior in a microfluidic or mesofluidic setting by reducing the surface tension in the containing solution. Passage through the valves present in the retaining walls in the cartridge is made more efficient by the surfactant. This allows for a shorter amount of time needed to centrifuge the density medium into the measurement chamber during manufacture. Most surfactants incorporate into hydrophobic cell membranes, causing destruction of the cell by lysis. Destruction of the cell membrane can prevent pelleting of the cells or cause inaccurate pellet heights. Therefore, a surfactant that does not destroy cells, such as Pluronic® surfactants, is an advantageous component of the density medium. Such surfactants may provide the further advantage of retarding evaporation by forming a monolayer at liquid-air interfaces.

[0015] Specifically claimed are density medium formulations and compositions as described above, in any combination.

[0016] The production of a distinct, sharp, and easily visible pellet opens up the potential for colorimetric assay applications. Colorimetric assays are known in the art; however, their application is used for homogeneous samples in combination with a spectrophotometer, or by staining and viewing a sample directly under a microscope. Both of these applications require specific technical training to provide an informative answer. In the context of home consumer use, heterogeneous pellets may be analyzed first by concentration using the cartridges, and then by condition with a colored or florescent dye. A dye or marker may be added to the sample or density medium that could indicate a particular condition of the contents of the pellet to the user. In some embodiments, it may be advantageous to determine cell viability in addition to cell concentration. For instance, the fraction of viable sperm cells may be measured in addition to the sperm concentration to help determine semen quality. It is known in the art that viable cells exclude certain dyes due to the presence of an intact membrane; examples are trypan blue or eosin-Y. Example dyes also include stains that create color only if the cell is living. For instance, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) creates a distinct color change upon reaction with metabolic products within living cells. If cells are incubated with such a dye prior to compaction, the color of the pellet will reflect the fraction of cells that are viable. For example, a cell population treated with an exclusion dye produces a more intensely colored pellet when containing a higher proportion of non-viable cells. Unbound dye may be separated from the cells following centrifugation by a density medium, enhancing the contrast between living and dead cells. In an embodiment, color may be measured by a smart phone or other camera which may include light sensitivity in the ultraviolet (UV), infrared (IR), or visible light spectra, or any combination thereof; the cartridge containing a dyed sperm pellet may be photographed in the presence of a card printed with color blocks matched to specific

concentrations of viable cells. Using automated color recognition, an application may display a quantitative (based on dye color intensity normalized by the use of the control color blocks printed on the card) or semi-quantitative (whether the result shows more or less than 60% viable cells in the pellet by comparing the pellet color to a color block on the card) result to a user. In another embodiment, a card containing the specific color intensities may be used to match cell viability percentage by un-aided visual comparison.

[0017] A density medium may comprise cesium chloride (a high density salt that increases cell density by migrating into the cells through potassium channels), sodium diatrizoate (a high density salt that increases cell density by causing water to migrate out of the cells thereby increasing the resolution between cells and other moderate density debris), a surfactant that does not cause cell lysis (to enhance fluid movement between the chambers), and silica nanoparticles that have covalently attached coatings to prevent salt dependent gel formation.

[0018] The disclosed methods, devices and kits are often presented in terms of devices and methods for assay of semen samples for fertility analysis. However, these examples are provided for the purpose of illustration only. Claimed methods, devices and kits may also be used with other fluids or samples, such as motor oil, manufactured or natural food, including biologically active foods such as beer, cheese and yogurt; soil or water; naturally occurring and manufactured or modified organisms, and blood.

[0019] Fig. 1 depicts an embodiment showing key structural elements related to assay of an analyte, including cavities and chamber. Shown is top view; however, the body of the cartridge is not shown. 10 identifies the arrangement of these elements within a cartridge body. 19 is a sample cavity, centered on a spin axis of the cartridge (not shown). 11 is a wall on the distal side of the sample cavity, separating the sample cavity 19 from the intermediate chamber 12. It may have the form of a partial annular ring. 13 is a wall on the distal side of the intermediate chamber; separating the intermediate chamber 12 and the metering chamber 15. It may have the shape of a partial annular ring. 14 shows a density medium, as a hatched pattern, within the narrow sedimentation column and extending part way into the metering chamber 15. The analyte fluid flows, during centrifugal operation, medially to distally, from the sample cavity 19 through the intermediate chamber 12 into the metering chamber 15. There, the analyte is separated due to the interaction with the density medium 14 (hatched pattern), the metering chamber 15 and the sedimentation column, shown also as 14 (narrow portion). The metering chamber may have a funnel shape that fluidly connects to the valves 16 on its medial side and fluidly connects to the sedimentation column 14 on its distal side.

[0020] Embodiments include a cartridge free of additional cavities or chambers fluidly connected other than those shown and described in this Figure. Embodiments are free of a dedicated reagent chamber. Embodiments are free of use of any reagent. Embodiments are free of a chamber and free of a path for the purpose of mixing analyte with a reagent. Embodiments include a fluid flow in a straight, non-serpentine path from the sample cavity towards the sedimentation column. Dyes and colorants are not reagents. Some embodiments include an enzyme in or on the interior surface of the sample cavity.

[0021] Key features shown in Fig 1 include three valves in the sample cavity wall 11 : two end valves 17 and one center valve 18. Additional key features include two valves 16 in the intermediate wall 13. These valves act to meter the analyte in its path during centrifugal operation, maintain the analyte in the sample cavity prior to centrifugal operation, and prevent evaporation of the density medium 14 between manufacture and use of the cartridge. Valves may be a depression in the height of the walls, although other forms or locations of valves may be used. Valve operation is discussed more elsewhere herein. Embodiments may have a different number of valves in walls than the number of valves shown and described in this Figure. Valves may be considered as or described as points of entry into the chamber on the distal side of the valve. Valves may be considered as or described as exit points from the cavity or chamber on the medial side of the valve. In one embodiment: valves 17 are at the far ends of wall 11; valve 18 is at the midpoint of wall 11; valves 16 are at the endpoints of wall 13; as shown.

[0022] In one embodiment, the capacity of the sample cavity is 250 microliters (μΐ _^ ); the capacity of the metering chamber is 250 μΐ.; the quantity of density medium is 80 μΐ.. The volume of the sedimentation column is small, such as between 1 and 15 μΐ.. Volumes of the cavity and chambers may vary considerably depending on application. A range of volumes may be the above stated volumes: +100% -50%; +50% -25%, +100% -75%, +100% -90%, +50% - 95%, in any combination.

[0023] In one embodiment the quantity of analyte recommended is the same as the volume of the sample cavity, such as 250 μΐ.. The quantity of analyte metered into the metering chamber may be the volume of the metering chamber minus the volume of the density medium, such as 250 - 80 μΐ., = 170 μΐ., for example. Excess analyte may remain in the intermediate chamber or the sample cavity, or both.

[0024] Fig. 2 shows the change in analyte fluid location in the cartridge during centrifugal operation. The initial state is before or at the start of centrifugal operation. The intermediate state is during spin up rotational acceleration. The final state is at the completion of centrifugal operation.

[0025] In the initial state, shown in the Figure, 19A shows the sample cavity filled with analyte, hatched. Reference designators 10 through 19 identify the same structural elements as in Fig. 1. 11 is the sample cavity wall. 13 is the intermediate chamber wall, 15 is the metering chamber. 14B, hatched, identifies the density medium, here shown in the sedimentation column and partially in the metering chamber 15. Valves 17 A, 17B and 18 are shown. Valves 16 are shown. A cartridge radial axis 27 (not the orthogonal spin axis (66 in Fig. 6), bisects the cartridge portion shown in the initial state in this Figure, vertically, 27.

[0026] In the intermediate state, the analyte 20, hatched, is forced both distally towards the sedimentation column due to centrifugal force and also off the cartridge radial axis 27 by radial acceleration, as shown. Because much of the analyte is off the radial axis it flows preferentially through valve 17B leaving valve 17A open for return air from the metering chamber 15 and the intermediate chamber 12. Having a valve for return air assists movement of analyte. Analyte fluid flows through valve 17B and possibly through valve 18 into the intermediate chamber, shown as 21, hatched. The analyte then flows from the intermediate chamber through one or both valves 16 into the metering chamber 15. The pair of valves 16 also operate as an analyte distal flow valve and an air medial flow return valve, similar to 17B and 17A, respectively.

[0027] The length of time for the intermediate state may be highly variable depending on application. For one embodiment this time is about 10 seconds. Such time may vary from 100 milliseconds to 10 minutes, or from 1 second to 30 seconds.

[0028] Centrifugal operation is needed for the pellet 29 to form as a result of the analyte and the density medium operating together. Such time may be 6 minutes. A range of times may be 1 to 60 minutes, or 3 to 15 minutes. The final state is shown at the completion of centrifugal operation.

[0029] For the final state, 28 shows the combined analyte and density medium in the sedimentation column and metering chamber 22 22, hatched, identifies fluid filling the metering chamber. Typically, layers are formed with the lighter analyte fluid on top (medial, top in the Figure) of the density medium, the density medium in the middle, and a sedimented pellet 29 at the bottom (distal, bottom in the Figure). The pellet 29 is shown, but other layers are not shown. Excess analyte, that is, analyte that does not participate in the interaction with the density medium 28 and does not contribute to the pellet 29 may be left in the intermediate chamber 12 or in the sample cavity 19B. Such possible excess analyte is not shown in the Figure. 19B in the final state shows an empty sample cavity. The pellet 29 may be a quantity of sperm from analyte comprising semen. Some embodiments identify motile v. non-motile sperm. The pellet may be white and may be readily visible to the eye, camera or sensor, particularly if the sedimentation column is between a clear and a pigmented (e.g., black) containment wall. Metering marks, not shown, may be used to provide a rapidly readable quantitative reading of pellet height.

[0030] Fig. 3 shows a side cross-section view and a top view of an embodiment of a cartridge 10. Reference designators 10 through 19 are the same as shown and described in Fig. 1, above. The cartridge in one embodiment is designed to contain a small volume of liquid (50 - 200 microliters, μΐ.) formulated to a density media to exclude less dense particles from compaction in the sedimentation column 14. To prevent evaporation, the cartridge 10 may be constructed from a moldable material with low vapor transmission properties, such as a cyclic olefin copolymer, one such example being Topas COC Polymers (from Topas Advanced Polymers, +1 (859) 746-6447, www.topas.com). The cartridge 10 also comprises a sample cavity wall 11 on the distal side of the sample cavity, separating the sample cavity from the intermediate chamber; and an intermediate cavity wall 13 between the intermediate chamber and the metering chamber. The sample cavity wall 11 comprises two or more points of entry 17 that behave as valves, regulating a controlled volume of analyte to pass during spin operation of the cartridge from the sample cavity to the intermediate chamber and then to the metering chamber while minimizing vapor transmission from a density medium to the sample cavity or out of the cartridge. One or more valves 17 also permit air to pass backwards from the intermediate chamber to the sample cavity, aiding the flow of analyte in the distal direction, as described elsewhere herein.

[0031] Fig. 3 also shows two or more points of entry 16 on the distal side of the intermediate chamber, separating the intermediate chamber from the metering chamber. These points of entry function as valves, regulating a controlled volume of analyte to pass during spin operation of the cartridge from the intermediate chamber to the metering chamber while minimizing vapor transmission from a density medium to the intermediate chamber or out of the cartridge. One or move valves 16 permits air to flow backwards from the metering chamber to the intermediate chamber, aiding the flow of analyte in the distal direction, as described elsewhere herein.

[0032] Walls 11 and 13, as well as points of entry and valves 17 and 16 have numerous configurations in embodiments, including a variable number of points of entry. A point of entry may be a depression in the top of the wall. It is advantageous to place two points of entry 17 in wall 11 at the two ends of wall 11. During spin acceleration analyte will accumulate more at one point of entry leaving the other open for air return. It is advantageous to place one point of entry 17 in wall 11 at the center point of the wall 11 to aid in clearing a controlled amount of analyte from the sample cavity. It is advantageous to place two points of entry 16 in wall 13 at the two ends of wall 13. During spin acceleration analyte will accumulate more at one point of entry leaving the other open for air return. It is sometimes advantageous to place one point of entry 16 in wall 13 at the center point of the wall 13 to aid in clearing a controlled amount of analyte from the intermediate chamber.

[0033] Fig. 3 shows both a side view cross-section and a top view of the cartridge, with certain chambers made visible, to show a construction embodiment of the cartridge.

[0034] A top portion 31 and a bottom portion 32 of the cartridge 10 form all or a bulk of the cartridge. These top and bottom monolithic portions are welded together using a method appropriate for the composite material, such as laser welding, ultrasonic welding, or adhesive. The top portion 31 of the cartridge 10 may contain metering marks 38 that visually show quantitative volumes of sedimented material in the sedimentation column 37. The top portion 31 of the cartridge 10 may be made of transparent material so a user can see into the sedimentation column 37. The bottom portion 32 in one embodiment is dark pigmented or colored black to allow contrast with of a view of a formed white or light colored column pellet, such as might be created by sedimented sperm cells. In another embodiment, metering marks 38 may be provided by a sticker, ink, paint or other markings placed on the outer surface of the top 31 of the cartridge, 10. Such a sticker may be opaque or dark over portions of the cartridge such that only the open sample cavity 36 and the sedimentation column (and optionally the metering chamber) 37 are visible as shown in the top view in this Figure. Such an element and embodiment provides clear, simple, reliable operation and use for an end-user, typically not trained medically and with no prior experience using the device. Dimensions, shapes and proportions in views in Fig. 3 are not to scale and are schematic only.

[0035] Monolithic construction, such as injection molding, of the top 31 and bottom 32 of the cartridge 10 is desirable, but not required. Embodiments include such a cartridge free of any other structural elements. A label, sticker or seal over the sample cavity is not considered a structural element of the cartridge. Density medium is not considered a structural element.

[0036] 33 shows a hub for attachment to a spin shaft. Ideally, this is part of the monolithic bottom 32 of the cartridge. 34 shows an opening in the hub that mates directly or indirectly with a spin shaft (not show). The hub 33 and opening 34 are discussed more elsewhere herein.

[0037] Fig. 4 shows schematically a mechanism and purpose of the density medium. A heterogeneous sample analyte (43 and 44) in a medial volume 41, containing a mixture of components of varying density, may be layered on a density medium, shown as hatched in both a medial volume 41 and distal volume 42. Centrifugal force during spin operation may act on the analyte causing particles denser 43 (shown as circles) than the density medium 42 to migrate through the density medium (hatched) from volume 41 to volume 42 while less dense particles 44 (shown as squares) are retained on medial (top) volume 41 of the hatched density medium. This principle is the basis for visually measuring or estimating the volume, weight or

concentration of particles with known density by the packed volume occupied by the denser particles 43. Packed particles 43 are a more theory-oriented representation of a pellet, such as 29 in Fig. 2. Embodiments are free of a reagent. Embodiments are free of nanoparticles. [0038] Fig. 5 shows a cross section of an embodiment of a cartridge 10 with a removable top seal 50 to further minimize evaporation of a density medium 14C. 53 shows the entire cartridge 10 optionally inside of a sealed package as indicated by 53, which further reduces evaporation and also protects the cartridge 10 prior to use. 10 identifies the complete cartridge with density medium 14C loaded and removable seal 50 placed on top. 52 shows the opening of the sample cavity 19, when seal 50 is removed. 11 and 13 represent walls as described above.

[0039] In some embodiments of the cartridge 10, density media may be placed in the metering chamber 14C prior to use. To allow for a suitable shelf life of the cartridge 10, some additional vapor transmission barriers may be required to maintain the density medium against evaporation. While the walls 11 and 13 provide some measure of vapor transmission protection, shelf life of the cartridge can be enhanced, especially when small volumes of density medium are concerned. As shown in Fig. 5, a removable seal 50 may be applied over the otherwise open top of the sample cavity 19. The removable seal 50 in one embodiment is composed of aluminum and adhered to the cartridge material around the sample cavity opening 52 by a pressure- sensitive adhesive with low vapor transmission properties. The aluminum may also be adhered to the cartridge by heat-sealing, removable adhesive, or other methods known in the art. Further protection may be afforded by enclosing the cartridge 10 in a pouch 53 or other secondary packaging that has an air and vapor tight seal. In one embodiment, the pouch 53 is composed, in part, of a layer of aluminum or other vapor-barrier material. Various combinations of walls 11 and 13, seals 50, and pouches 53 may be used to extend the shelf life of a cartridge containing a liquid in storage, such as a density medium, and increase commercial viability of producing large lots of the cartridge. 54 is a bottom of the cartridge. 55 is a top of the cartridge. When the top 55 is permanently attached to the bottom 54, one embodiment of a cartridge is created.

[0040] Fig. 6 shows an additional side cross-section of an embodiment of the cartridge and a top view of selected chambers, schematically. Fig. 6 shows selected fluid as hatched. The combined density medium and analyte is shown after spin operation as 61, hatched.

[0041] Operation of the cartridge to perform an assay of an analyte requires high-speed spin for centrifuge effect. An embodiment of the cartridge consists of the entire spinning assembly in operation, not including a motor and spin shaft. As such, it is desirable that the cartridge be well balanced about its spin axis 66. In one embodiment the cartridge has two arms, shown in Fig. 6 generally as 62 and 63. The analyte and density medium, after centrifugal operation, are shown in the metering chamber and sedimentation column, hatched 61, in cartridge arm 62. Cartridge arm 63 performs the function of counterweight and aerodynamic balance. Because of the weight of the analyte and the density medium 61, cartridge arm 62 is manufactured, prior to the addition of a density medium, as lighter than arm 63. Ideally, the cartridge would be balanced under all spin conditions, including at startup and at completion of centrifuge operation. However, since analyte is centered in the sample cavity on the spin axis 66 initially and then entirely or partially in arm 62 during and at the end of centrifuge operation, it is not simple or convenient to maintain perfect cartridge balance under all conditions.

[0042] From an aerodynamic point of view, the two arms 62 and 63 should have equal external shape, be as thin as possible, and be shaped to minimize drag. To achieve balance, arm 63 should be manufactured to be heavier than empty arm 62, yet have the same external cross section. Although there are no valves in the sample cavity wall for arm 63, the mass of these valves is very small. Therefore, additional material, such as molded plastic, may be included internally in arm 63. Such additional material may include a wall (not shown) inside the cavity 65 in arm 63. Note that arm 63 may also have a mirror of wall 13 shown in Fig. 2. If such a mirror image wall is not included in arm 63, additional weight in arm 63 must also compensate for this missing wall. Additional material in arm 63 may be used in the mirror image of the metering chamber (or elsewhere in the arm) in a variety of ways, such as thicker walls, thicker top and bottom, posts, walls or other shapes. It is generally not desirable to lump the additional weight in a single location, as such thicker plastic may interfere with optimal injection molding. It is also desirable to not add the weight near the distal end of the arm 63 as this increases the time required for spin up, reduces battery life, and could add to aerodynamic instability.

[0043] Fig. 7 shows an embodiment of the device for estimating concentration of cells 71 based on volume occupied by cells 71 in a packed volume. The cells 71 are initially suspended in a fluid 72. In one embodiment, the cells 71 are sperm cells and the fluid 72 is seminal fluid. Following centrifugal rotation, cells 71 are packed at the bottom of a sedimentation chamber 73 and the sedimented cells occupy a volume proportional to a number of cells 71 initially suspended in the fluid 72.

[0044] Fig. 8 is a flowchart of an embodiment of a method for using devices described herein and a method of estimating or quantitative measuring of sperm count based on volume occupied by sperm cells packed into a sedimentation column of defined cross-section following centrifugation. In one embodiment, the method is performed using a kit comprising a collection cup, a cartridge, a transfer device such as a pipette or dropper and an instrument to provide centrifugal operation on the cartridge.

[0045] The user collects 80 a sample or fluid in the collection cup. In one embodiment, the collection cup comprises digestive enzymes such as chymotrypsin, trypsin, bromelain, or papain for accelerating liquefaction of the fluid. The fluid is swirled or agitated 81, for example by the user, in the collection cup (or the sample can be otherwise agitated, such as agitated by the instrument once it is placed in the instrument). Swirling or otherwise agitating the fluid accelerates dissolution of the enzyme into the fluid. A first interval of time (such as 1 to 120 minutes, or 2 to 30 minutes) elapses to allow the enzyme to liquefy the fluid. A portion of the fluid is then transferred 82 to the cartridge using a transfer device, such as a pipette, dropper, syringe or bulb transfer pipette. Ideally, the pipette is calibrated, such as holding a fixed amount of fluid, to enable the user to easily and reliable transfer a correct quantity of fluid from the collection cup to a cartridge. In one embodiment, the cartridge is capped with a lid or sticker following placement of the fluid, such as in a sample cavity of the cartridge. The cartridge is attached 83 to a spin shaft of an instrument, as described elsewhere herein. Optionally, the instrument may accelerate the cartridge in one direction and then an opposite direction for an interval of time, mechanically agitating the fluid, encouraging homogenization and reduced viscosity for more consistent measurements. The instrument may also accelerate the cartridge in one direction, allow it to come to a stop, then repeat for an interval of time to provide mechanical agitation. The instrument spins 84 or rotates the cartridge at a rotation rate for a second time interval (e.g., for 2-10 minutes at 2000-10000 RPM). The method may employ a controlled spin acceleration rate, as described elsewhere herein. Such an acceleration time may be in the range of 1 to 60 seconds, or in the range of 2 to 15 seconds. Optionally, the cartridge is spun at a reduced rotation rate for a third interval of time (such as for 0.5 to 5 minutes) to allow for controlled expansion of compacted cells in a sedimentation column of the cartridge. After centrifugal operation is complete at the end of the second time interval, the cartridge rotation is automatically terminated. The user then removes the cartridge from the instrument 85. This step 85 is optional, as the cartridge may be readable in the instrument. In step 86 the user reads the result as described elsewhere herein. [0046] In some embodiments, the instalment comprises a digital reading the user can read (e.g., digital reading on a user interface of the instrument). All embodiments of the instrument described herein may comprise a digital reading on a user interface of the instrument. In one embodiment, the instrument comprises a lid, wherein the lid comprises one or more magnets and the instrument comprises one or more sensors configured to detect magnetic fields. The one or more magnets and one or more sensors are placed within the lid and the instrument respectively, such that, when the lid is closed on the instrument, the one or more magnets in the lid and the one or more sensors in the instrument are a distance away from each other, where the distance is less than a threshold distance necessary for the one or more sensors to detect a magnetic field of the one or more magnets in the lid and thus detect that the lid is closed on the instrument. In another embodiment, the one or more magnets can be in the instrument and the one or more sensors in the lid of the instrument. The magnet and sensor configuration described here can be applied to any instrument described herein.

[0047] Another exemplary method, not shown in Fig. 8, allows a user to perform a controlled test using a control, calibrated, test or practice sample or solution. For this method, steps 80 through 82 are replaced by the following steps: (i) open a container containing the control, calibrated, test or practice sample or solution; (ii) transfer fluid from this container, using a calibrated pipette to a cartridge. Steps 83 through 86 are the same. An additional step, after step 86 is: (iii) compare the read value on the cartridge to an expected value for the control, calibrated, test or practice sample or solution.

[0048] Yet another exemplary method is to first perform the controlled test method described above, and then perform the steps in Fig. 8 using a new cartridge.

[0049] A user may perform steps 80, 82, 83, 85 and 86 manually, himself or herself, without assistance of a medical professional. An instrument may perform automatically some or all of steps 81 or 84, or both. An instrument may provide some or all timing functions and a spin function. Any number or combination of steps may be performed by a medical professional.

[0050] In some embodiments an enzyme is in a sample cavity and a collection cup is free of enzyme, which may permit step 81 to be eliminated from a claimed method. As discussed elsewhere herein, an automated or semi-automated instrument may provide treatment of collected fluid with an enzyme, such as by providing spin or other agitation. Thus, the equivalent purpose of step 81 may be performed after step 83. Step 85 is optional, as a pellet height may be read while the cartridge is still attached to an instrument. Step 86 may be partially or fully automated. Step 82 may be unnecessary in some method embodiments. Effective fluid volume may be obtained by filling the sample cavity to a directed level.

[0051] Fig. 9 shows an enlargement of a top view and a side view of a sedimentation column, the sedimentation column comprising metering marks 92 and quantitative numbers 93. After cells are compacted by centrifugation, the height of a resulting pellet 94 may be determined visually by differences between cells in the pellet 94 and fluid 95 or by other means including fluorescent cell labels. A user may estimate initial concentration of cells in the fluid by reading the number 93 closest to a metering mark 92 closest to the interface between the cells 94 and the fluid 95.

[0052] Fig. 10 shows an enlargement of a top view and a side view of a sedimentation column, the sedimentation column comprising metering marks 102 and numbers 103. The sedimentation column comprises a lens 106 configured to magnify the sedimentation column and size of the sedimentation column. The lens 106 can be integrated into the sedimentation column during fabrication, for example, by injection molding of polymer. The presence of the lens 106 may allow the user to visualize an interface between a pellet 104 and fluid 105 more easily. In one embodiment, the lens 106 is cylindrical in shape. Other types and shapes of lenses can also be used. After cells are compacted by centrifugation, the height of the pellet 104 may be determined visually by differences between the cells and fluid 105 or by other means including fluorescent cell labels. The user can estimate the initial concentration of cells in the fluid by reading the number 103 closest to a metering mark 102 closest to the interface between the cells 104 and the fluid 105.

[0053] Fig. 11 shows an alternate embodiment of a sedimentation column intended for use in estimating a wide range of cell concentrations. The sedimentation column comprises course metering marks 111 and fine metering marks 112. After cells are compacted by centrifugation, height of a resulting pellet 113 may be determined visually by differences between the cells in the pellet 113 and fluid 114 or by other means including fluorescent cell labels. The user may estimate initial concentration of cells in the fluid by reading a number 112 or 111 closest to an interface between the cells in the pellet 113 and the fluid 114. In this embodiment, the sedimentation column is tapered comprising a section of a low cross-sectional area 115 and a high cross-sectional area 116 with a transition area 117 in between the sections 115 and 116. In this embodiment, a pellet comprising low cell concentration will be measured by a portion of the sedimentation column comprising low cross-sectional area 115, while a pellet comprising substantially higher cell concentrations will be measured by a portion comprising high cross- sectional area 116. The spacing of calibrated metering marks 111 and numbers 112 are set in relationship to the different cross-sectional areas, allowing a user to accurately estimate cell concentration over a wide range. To one skilled in the art, it is apparent that many variations of sedimentation column taper are possible. For instance more than one transition area 117 may be integrated into the sedimentation column to create multiple sections with varying cross-sectional area. For example, the multiple sections may comprise sections with sequentially increasing or decreasing cross-sectional areas. In another example, cross-sectional area may continuously increase or decrease along the sedimentation column, as in a taper, to accommodate a wide range of cell concentrations and spacing of metering marks 111 and numbers 112 is set accordingly. The multiple sections may also comprise varying cross-sectional areas such that a visual of the height of the pellet 113 in the sedimentation column corresponds to cell concentration of the pellet 113. Marking lines and marking quantity number may not be one to one.

[0054] Fig. 12 illustrates an example of a sedimentation column of a fluid following centrifugation, wherein the fluid comprises particles or materials with a density higher than the fluid's density but lower than density of certain cells or particulates in the fluid. Following centrifugation, the intermediate density particles or materials form an intermediate layer 124 between the pellet 125 of compacted cells and the fluid 126. The intermediate layer 124 may comprise distinctively colored particles or materials such as dyed polystyrene or another polymer in order to enhance optical contrast of an interface between the pellet 125, intermediate layer 124, and fluid 126. Markings 123 and 122 may be used to provide a quantitative determination of the volumes of particles in layers 125 and 124, and potentially 126.

[0055] Fig. 12 also schematically represents an embodiment of the sedimentation column of a fluid following centrifugation, wherein the fluid was mixed with a dye prior to centrifugation that identifies dead cells. For example, the dye can selectively partition into dead cells but not living cells. It is known in the art that dead or immotile sperm cells have a density lower than the density of living and motile sperm cells. The sedimentation column typically comprises or is proximal to reading marks, which may or may not be quantitative, such as marks 123 and 122. Following centrifugation, the fluid separates into layers with a fluid layer 126 closest to the center of the centrifugation, a live cells layer or a pellet 125 furthest from the center of the centrifugation, and a dead cells layer 124 with intermediate density in between the two layers 126 and 125. Living cells exclude the dye and therefore are visually distinct from the dead cells layer 124 and fluid layer 126 which also exhibit the color of the dye. The user may also estimate number of dead cells from the visually distinct dead cell layer 124. As described previously, an intermediate density layer formed from polymer fragments or particles may be mixed into the fluid prior to centrifugation to enhance the contrast between the dead cells layer 124 and pellet 125.

[0056] Figs. 13 and 14 illustrate various embodiments of mechanisms for attaching a cartridge to a spin shaft of an instrument. These embodiments may be used with any of the cartridges or instruments described herein, or may be used to attach the cartridge to other instruments outside of those described herein. In an embodiment, the cartridge, the motor shaft or spin shaft or an adaptor configured to attach to the motor may comprise a magnetic material, providing a mechanism for attaching the cartridge to the motor.

[0057] Fig. 13 shows an embodiment of a schematic for attaching a cartridge 131, a portion of which is a hub, is shown 134. The hub 134 comprises a cavity 133, such as a hollow cylinder open on one end. The cavity 133 comprises a first diameter less than or equal to a second diameter of a shaft 134 of the motor 132. To attach the cartridge to a spin shaft 134 powered by a motor 132, the receiving cavity 133 in the cartridge hub 134 is friction fit or press-fit with the spin shaft 134. Material used in the first diameter of the cavity 133 and elastic modulus of material used for the cartridge hub 134 may be selected such that a suitably tight friction fit for an end user is established. Those in the art know that many shape variations are possible for both the spin shaft 134 and the mating cavity 133. For example, the spin shaft 134 may comprise a keying flat, or be hexagonal, or have a retaining lip or retaining groove. The mating cavity 133 may be round, square, hexagonal, or have internal crush ribs. It may have elements to mate with the shaft's keying flat, hexagonal shape, or retaining lip or groove. One or more magnets may be used in place of or in addition to a friction fit. For example, a magnet, which may be press fit into cavity 133, may provide a more consistent mating and un -mating force and one that will not change with extensive reuse. A magnetic mount may also provide an end user with no or little experience with a positive mating feedback haptic to assure simple, proper, reliable, confident and safe operation. [0058] Fig. 14 shows another embodiment of a schematic for attaching a cartridge 141 to a spin shaft 145 of a motor 142. The cartridge hub comprises a cavity 143. The cavity 143 comprises a shape and the adaptor 144 comprises the same shape and is configured to fit in the cavity 143. The adaptor 144 is attached to the spin shaft 145. To attach the cartridge hub to the motor 142, the adaptor 144 is press-fit into the cavity 143. Material used in the first diameter of the cavity 143 and elastic modulus of material used for the cartridge 141 may be selected such that a friction fit is established between the spin shaft 145 and cavity 143 of the cartridge 141 allowing rotation of the cartridge 141 when attached to an instrument. The material of the adaptor 144 may comprise notches configured to allow the adaptor to flex creating a secure fit between the cartridge 141 and adaptor 144. In another embodiment, the motor 142 may comprise a cavity-containing socket and the cartridge 141 may comprise a corresponding projection. Additional projections, ribs, recesses or detents may be added to the adaptor 144 in order to create a "snap" fit with the cartridge 141. Exemplary ribs are shown in the bottom view in the Figure.

[0059] Fig. 15 illustrates an embodiment in which a liquid medium of defined density is used to separate particulates based on unique physical characteristics of the particulates. In one embodiment, the cartridge 151 is loaded with a volume of a density medium 152; the density medium 152 comprises a fluid medium of a defined density. The density medium 152 occupies a defined volume of a sedimentation column 153 integrated into the cartridge 151. The size, shape and material of the sedimentation column, the metering chamber, and optionally the intermediate chamber and sample cavity as described elsewhere herein are adapted to be able to hold the density medium. The density medium may be stored within a cartridge, part of a claimed manufactured device or method, or included as part of a kit. The sample fluid is loaded through a sample cavity 154 of the cartridge and the cartridge is spun at a specified rotation rate for an interval of time such that a defined volume of the sample fluid initially layers upon the density medium 152 in the metering chamber and sedimentation column 153. During centrifugation, particulates in the sample fluid that comprise a higher density than density of the density medium 152 will sediment to the end of the sedimentation column during centrifugation, forming a pellet 155. The height of the pellet 155 may be measured to estimate initial concentration of higher density particulates as described previously. Excess fluid and particulates comprising a density less than density of the density medium will remain suspended as a supernatant 156. The sample fluid may comprise semen, and the particulates may comprise sperm cells. The unique physical characteristics of the sperm cells may comprise a density, the density characteristic determined by cell motility, viability, or morphology. In some

embodiments, the density medium may comprise a fluid of specified density configured to separate sperm cells from other particulates found in semen such as cell fragments and leukocytes (i.e. the density medium is less dense than the sperm cells and denser than the other particulates). In this embodiment, the pellet 155 may be measured to estimate the concentration of sperm cells without interference from other particulates in semen. In some embodiments, the density medium 152 may comprise a fluid of specified density configured to separate motile from non-motile sperm cells (i.e. the density medium is more dense than non-motile sperm cells and less dense than motile sperm cells). In this embodiment, the pellet 155 may be measured to estimate the concentration of motile sperm cells. In another embodiment, the density medium 152 may comprise a specified density configured to isolate X-chromosome containing sperm cells from Y-chromosome containing sperm cells (X-chromosome containing sperm cells are on average denser than Y-chromosome containing sperm cells).

[0060] The element 153 in the Figure may be a portion of a sedimentation column; all of a sedimentation column, or a sedimentation column and a portion or all of a metering chamber. The fixed width shown in the Figure is non-limiting with respect to different shapes, tapers, cross-sectional areas and volumes.

[0061] Note that the terminology of "top" and "bottom" with respect to a sedimentation column is with the sedimentation column oriented vertical, with a sedimented pellet at the bottom. However, in use, a cartridge is horizontal and so "top" and "bottom" with respect to the cartridge as a whole and with respect to the sample cavity use this orientation.

[0062] The terms, "cavity" and "chamber" are used interchangeably, unless otherwise clear from the context. The term, "cavity" is preferred for clarity for a volume that is generally open, such as at its top. The term "chamber" is preferred for clarity for a volume that is generally closed except via specific entry and exit points.

[0063] A dyed sperm pellet or other dyes or colorants are not to be construed as reagents. They do not typically combine chemically with an analyte.

[0064] Analyte and density medium describe specific composite substances, which may be, but not necessarily, fluids, gels, pastes, slurries, emulsions, or suspensions in any combination. In centrifuge operation they move primarily as fluids or a fluidic mixture. References to a "fluid" should be construed broadly in this context.

[0065] Elements of a completed cartridge may comprise a monolithic bottom, a monolithic top, a cover sheet, a seal, density medium and an envelope. The cover sheet, seal, and envelope are individually optional. A cartridge adapted to accept but not yet comprising a density medium is specifically claimed. The top and bottom need not be monolithic and non-monolithic construction should be construed using the doctrine of equivalents. Other shapes, sizes and orientations of arms, motor attachment, chambers, walls, valves and sedimentation column, if they perform the same function as described in a claim, should be construed using the doctrine of equivalents. A cartridge may alternatively be called a prop. An instrument may alternatively be called a centrifuge.

[0066] Specifically claimed are cartridges, methods of use of a cartridge, methods of manufacturing a cartridge, methods of assay using a cartridge, and a kit of components including a cartridge sufficient to perform some or all of method steps disclosed. Specifically claimed are kits both with and without a control solution and a cartridge for performing a control of method steps. A control solution may contain a known quantity or concentration of particles that will create a pellet under use. Specifically claimed is one or more cartridges with an instrument to provide spin. Claims specifically include cartridges both with and without a density medium, wherein the cartridges are adapted to accept a density medium as disclosed. Claims shall be construed and components for cartridges or methods shall be measured (e.g., "quantity") relative to unique value or inventive step contribution to a device or method. Claims specifically include cartridges both with and without markings.

[0067] Ideal, Ideally, Optimum and Preferred— Use of the words, "ideal," "ideally," "optimum," "optimum," "should" and "preferred," when used in the context of describing this invention, refer specifically a best mode for one or more embodiments for one or more applications of this invention. Such best modes are non-limiting, and may not be the best mode for all embodiments, applications, or implementation technologies, as one trained in the art will appreciate.

[0068] All examples are sample embodiments. In particular, the phrase "invention" should be interpreted under all conditions to mean, "an embodiment of this invention." Examples, scenarios, and drawings are non-limiting. The only limitations of this invention are in the claims. [0069] May, Could, Option, Mode, Alternative and Feature— Use of the words, "may," "could," "option," "optional," "mode," "alternative," "typical," "ideal," and "feature," when used in the context of describing this invention, refer specifically to various embodiments of this invention. Described benefits refer only to those embodiments that provide that benefit. All descriptions herein are non-limiting, as one trained in the art appreciates.

[0070] All numerical ranges in the specification are non-limiting examples only.

[0071] Embodiments of this invention explicitly include all combinations and subcombinations of all features, elements and limitation of all claims. Embodiments of this invention explicitly include all combinations and sub-combinations of all features, elements, examples, embodiments, tables, values, ranges, and drawings in the specification and drawings. Embodiments of this invention explicitly include devices and systems to implement any combination of all methods described in the claims, specification and drawings. Embodiments of the methods of invention explicitly include all combinations of dependent method claim steps, in any functional order. Embodiments of the methods of invention explicitly include, when referencing any device claim, a substation thereof to any and all other device claims, including all combinations of elements in device claims.