Fluid delivery system for a flow cytometer

Abstract

Systems for sheath fluid and sample fluid transport from containers to an analytic device, such as a flow (cell) cytometer, are disclosed. These systems are sterile and remain so, as they are closed to the ambient environment. As a result, fluids of high purity reach the analytic device, and in the case of a flow cytometer, the desired cells are sorted in sort of high purity.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is related to and claims priority from commonly owned U.S. Provisional Patent Applications: Ser. No. 60/476,380, filed Jun. 6, 2003, entitled: TUBING SYSTEM FOR USE WITH A CELL CYTOMETER, Ser. No. 60/504,105, filed Sep. 19, 2003, entitled: SHEATH DELIVERY SYSTEM, and Ser. No. 60/526,747, filed Dec. 3, 2003, entitled: SAMPLE FLUID DELIVERY SYSTEM, all three of these U.S. Provisional Patent Applications are incorporated by reference herein.

FIELD OF INVENTION

The present invention relates to systems, apparatuses, and methods, for use in sorting cells, whereby the inventions are used with any of a variety of flow cytometers. In particular, the present invention is directed to sterile systems for supplying and delivering sheath and sample fluid to the flow nozzle of a flow cytometer.

BACKGROUND OF INVENTION

Flow cytometry is a technology that utilizes an instrument in which particles, such as cells, in suspension and stained with fluorescent probes (for example, dyes), are passed single file in a fluid stream, and contacted by a laser beam. Fluorescent signals are emitted when the laser excites the probes, with the signals electronically amplified and transmitted to a computer. The flow cytometer sorts the particles, having specified properties, into collecting vessels, with the selected properties indicated by the specific probes. A computer communicates with the flow cytometer to cause the particles to be separated and directed to the selected populations. Resultingly, particles, such as human chromosomes or stem cells, can be sorted to a purity of about 99%, regardless of the composition being sorted. As such, flow cytometry, including Fluorescent Activated Cell Sorting (FACS), is a common method used to isolate various types of cells, including stem cells. Not only are cell sub-populations sorted, but data on the various cell populations are collected. As such, FACS allows for collection of information on the cell populations and sorting the population into discrete cell populations.

Flow cytometers move cell samples through the laser by use of two pressurized fluids, sheath fluid (also known as “sheath”), and sample fluid. Under differential pressures, together the fluids deliver the cell sample to the detection area. Precise delivery of the cells is required, as each cell must be contacted by a laser or lasers. After delivery to the detection area, also known as the laser/stream intercept, the cells in the sample fluid are individually separated in an aerosolized sheath, electrically charged, and separated into discrete populations as the cells pass through a magnetic field.

A conventional FACS sorter system 1 is shown in FIG. 1. In this system 1, cells are suspended in a sample fluid, initially held in a vessel 2, such as a polyethylene 5 ml test tube. The sample fluid is driven or forced to exit the tube by an external source of pressurized gas 2a, delivered through line 2b. The pressurized gas physically contacts the fluid in the vessel 2 and drives it into a line 2c and through to a flow nozzle 3 of a flow cytometer. The gas typically diffuses into the fluid, as a result of the physical contact. Similarly, the sheath fluid, typically held in a vessel 4, for example, an 8 liter stainless steel tank, is also driven by pressure, from an external source of pressurized gas 4a, and delivered through a line 4b. The pressurized gas physically contacts the sample fluid in the vessel 4a. The pressure causes the fluid to exit the vessel 4 through the internal tube 4c and to enter a line 4d, that connects to line 4e, through which the sample fluid reaches the flow nozzle 3 of the flow cytometer. Line 4d, through which the sheath fluid travels, normally includes a bacteria separating filter, such as a 0.22 micron (pore size) filter. But if particles in the sheath fluid are smaller than the pore size, they pass through the filter and contaminate the sample fluid at the flow nozzle. As a result, it is desired to have a system that ensures a higher likelihood that the fluid will be contaminate free. One way to prevent contamination is to limit contact with outside sources. Also, it is desired for the system, through which the sheath fluid flows, to be of a character whereby it can be sterilized.

The physical contact of the gas with the fluid typically results in the gas diffusing into the fluid. This is problematic because the character of the cells being examined can be altered. For this reason, it is preferred if the gas does not contact the sample.

Sheath fluid contaminated by microorganisms, microparticles, or nucleases, adversely impact the results of a study. More importantly, if stem cells are sorted for therapeutic uses, the cells are unavailable for use if contaminated. A common cause of contaminants in the sheath or sample fluid results from the introduction of contaminants by pressurized gas physically contacting the sheath or sample fluid. The gas potentially delivers dust particles or bacteria to the fluid. Current methods for preventing contamination involve placing bacteria catching filters along the sheath fluid lines, whereby the contaminating particles are removed. The filters remove bacteria of a particular size, but are ineffective should bacteria or other contaminates pass beyond the filters. As such, it is desired to have a system where introduction of bacteria and particles is inhibited.

Another way in which contaminants enter the system is through the waste lines 5 (FIG. 1). Waste line(s) 5 join to the system 1 at the sheath fluid line 4d, at a T-connector 5a, which includes a two-way manually actuated valve, for controlling the fluid flow between the feed line 4e and the waste line 5. Waste is transported over the waste line 5 to a waste tank 6, typically pulled by a vacuum, and/or pushed by pressure originating in the sheath tank. Additionally, the tubes used for transporting the sheath or sample fluid may not have been sterilized. Not only does the gas contain contaminants, but the gas typically diffuses into the sheath and sample fluid, affecting the purity of these fluids.

Typically, the components of the system are disassembled and washed with strong detergents. These washing procedures are elaborate, time consuming, and labor intensive. Also, some components, such as the filter, are not cleansed because exposure to detergents destroys the filter. Accordingly, such a system is not certifiably clean.

Applications of FACS are ideally performed under aseptic conditions so that isolated cells can be successfully cultured, transplanted, or processed for the isolation of nucleic acids. The currently accepted fluidic system design for flow cytometers is sub-optimal, because an aseptic environment for FACS is not provided. Hardware components directly contacting the sample and other components carrying fluids, which contact the sample, are not easily replaced or autoclaveable. To use FACS technology in the clinical setting, aseptic design changes are required. The current flow cytometry technology cannot be used for clinical sorting applications because the fluidic system is not designed for this intent. Both ends of the fluidic system are open ports for microorganisms, microparticles, and nuclease infection. Further, the contemporary tubing systems are not easily replaced with new sterile components.

The contemporary systems also experience changes in hydrostatic, or head, pressure, causing instability of stable aerosol formation. This is especially true during long sorts, such as those lasting four or more hours. Hydrostatic pressure changes, normally decreases in hydrostatic pressure, occur as the tank (or vessel) holding the sheath fluid goes from full to empty. This is in accordance with Torricelli's Law, as applicable to fluid height in a column (approximately 27.7 inches of water in a column equals 1 pound per square inch (psi)). Specifically, as sheath fluid is pushed from a lower elevation in the tank, the fluid must travel further upward to the flow nozzle. The increased vertical distance results in hydrostatic pressure decreasing at the flow nozzle. The effect of hydrostatic pressure is illustrated in FIGS. 2A and 2B. Initially, a fluid stream 7, from an opening 3a in the flow nozzle 3, that ends in a last attached drop (LAD) 8, is electrically charged based on known time intervals. In these figures, an electric charge is provided at a predetermined time interval (p), this time interval being constant and corresponding to the fluid stream 7 having traveled a distance D from the intercept (i) of the laser 9 and the fluid stream 7. This distance D is a calibrated distance, in accordance with the time interval p. As shown in FIG. 2A, charging is occurring under conditions of constant hydrostatic pressure. At time interval p, a particle of interest, such as a cell (c) is located in the LAD 8. When an electric charge is applied to the fluid stream 7, the charge is transferred to the LAD 8, immediately prior to the LAD's detachment, and the LAD 8 containing the cell (c) is charged. This is because the LAD 8 of the fluid stream 7 is at the distance D from the laser-fluid stream intercept (i) (corresponding to the calibrated distance of travel for the fluid stream 7 at time interval p). Once beyond this distance D, the LAD 8, with the cell (c) of interest therein, detaches from the fluid stream 7, and proceeds to be sorted by electromagnetic forces from the sorting plates 10. In FIG. 2B, the fluid stream 7 is under decreased hydrostatic pressure. This decreased hydrostatic pressure causes decreased surface tension, whereby the LAD 8, containing a cell (cx), of the fluid stream 7 occurs sooner, than would the corresponding LAD 8 when the fluid stream 7 is under constant hydrostatic pressure. Accordingly, the LAD 8 is at a distance d, which is less than the corresponding calibrated distance D, by a distance A. The LAD 8b containing the intended cell (c) is detached from the fluid stream 7 beyond the distance d. The LAD 8b, by virtue of its being detached at the time of charging, coupled with the distance A away from the LAD 8, receives an insufficient charge, and in most cases, receives no charge at all. Accordingly, the LAD 8b will go unsorted. The LAD 8 and the particle, for example, a cell (cx) of non-interest, therein, that was electrically charged (with the charge intended for cell (c) of LAD 8b), will be sorted by the sorting plates 10; however, since the cell (cx) therein is a non-intended particle, the sort purity is reduced. By reducing the sort purity of the collected cells, overall purity of the collected cells may be such that the entire FACS process is rejected.

Different pressures driving the sample fluid and the sheath fluid result in laminar flow conditions at the flow nozzle of the flow cytometer. As such, individual droplets exiting the flow nozzle, are composed of both sample fluid and sheath fluid. Thus, a constant pressure is desired.

As such, it is desired to have a system that can provide a sterilized environment. It is further desired to have a system that eliminates concerns associated with laminar flow.

SUMMARY OF INVENTION

The present invention relates to fluid delivery systems, for the maintenance and transport of both sheath and sample fluid to an analytic device. The systems provide sterile pathways, closed to the ambient environment, for both the sheath and sample fluid as they are delivered to a flow cytometer. Moreover, these sterile systems are formed from sterile subsystems, including tubing sets for the transport of sheath fluid, a bladdered tank for release of sterile sheath fluid, a sterile waste subsystem, and a sample fluid maintenance and delivery subsystem. The subsystems are also adaptable to mounting on platforms, including those with table mounted brackets.

The tubing system, upon its connection to a FACS sorter, is closed to the ambient environment. The tubing system may be unitary in structure, formed of molded components, to be a single piece. The tubing system facilitates sheath fluid transported from a source to a flow cytometer, whereby air bubbles are inhibited from forming in the system. The system is sterile, thereby eliminating the need for bacteria removal filters along fluid lines (tubes), due to the system being sterile and closed to the ambient environment. This system also eliminates the need for in-line valves along the system. The tubing system can include one or a plurality of tube members. The multiple tube members are used to remove gas from the lines. Also, the sheath and sample fluids are in bags that maintain sterility and where gas is not commingled.

The sheath fluid is transferred to the flow nozzle of the flow cytometer by the tubing system. Fluid is transported through the system without any in-line valves or in-line flow control devices. The tubing system is typically transparent or translucent, allowing the user to see the fluid flow therein and detect any turbulence, air bubbles, or other conditions within the system that destroy the stability of aerosol formation after the fluid exits the flow nozzle. By viewing the flow, the user has visual knowledge of changes in the flow and can control it instantaneously.

The present invention provides a sterile and disposable tubing system for FACS sorters, that meets requirements of high-speed FACS and GMP. This sterile tubing system includes a multitude of components.

The tubing system or set, through which the sheath fluid is transported, is universal and can be fitted to a variety of flow cytometers without significant modifications. It is designed to couple with a fluid source, an analytic device, and a waste system, and provide a sterile environment for the fluid. The tubing set is such that it couples easily and securely with a fluid source, analytic device, for example, a flow cytometer, and a waste system. The tubing set is also such that it uncouples easily with the aforementioned components. Additionally, the tubing set can be fit into prearranged configurations, such as table mounts and preconfigured waste systems. The tubing system is universal in that it can easily be fitted, placed onto, and removed from numerous flow cytometers without significant modifications. It also provides a path for the fluid that is closed to the ambient environment.

The present invention provides a sheath fluid delivery system that eliminates changes and fluctuations, typically decreases, in hydrostatic pressure at the flow nozzle of a flow cytometer. The present invention provides a tank that releases sheath fluid at a constant height, to travel a constant distance to the flow nozzle, to maintain constant hydrostatic pressure at the flow nozzle. This allows aerosol formation to be constant, resulting in highly stable FACS sorts.

The present invention also relates to a fluorescent activated cell sorting sheath delivery system (FSDS), that includes a bladdered sheath fluid source, for example, a bladdered vessel, and a closed, typically sterile and disposable, tubing system.

A sterile system for handling and transferring sample fluid to the flow cytometer is provided. The sample fluid system is a closed system, closed to the ambient environment, whereby the sample fluid is not in contact with airborne contaminants. The sample fluid system also includes a sterile reservoir and an apparatus for maintaining sterility of the sample fluid. Sample fluid moves from the sterile reservoir to the flow cytometer by pressure from a pressurized gas pressing the sterile reservoir, for example, a prepackaged sterile bag of fluid. The pressure causes the fluid to flow from the reservoir to the flow nozzle of the flow cytometer along a pathway that is not in physical contact with the pressurized gas. As a result, the sample fluid remains sterile from the reservoir to the flow cytometer.

The sample fluid system lacks any in-line valves, and includes sterile tubing that is removable, replaceable and autoclavable.

An embodiment of the invention is directed to a system for sheath fluid transport. The system includes a molded tubing set for coupling a source of sheath fluid, a waste system, and a flow nozzle. The tubing set includes a plurality of flexible tube members which may transport fluid under 120 psi, the tubing set is configured for providing a sterile environment for fluid transport from the fluid source to the flow nozzle.

Another embodiment of the invention includes a system for transporting sheath fluid to a flow nozzle of a flow cytometer. The system includes a tubing set for coupling with a container of sheath fluid, a waste system, and the flow nozzle. The tubing set has a plurality of flexible tube members, which may transport fluid at pressures of up to approximately 120 psi. The tubing set is such that it provides a sterile environment for sheath fluid transport from the container to the flow nozzle and the waste system. There is also a pressurizable tank for holding a container of sheath fluid, the tank including at least a first port for receiving pressurized fluid for driving the fluid in the container through the tubing set, and a second port where at least one flexible tube member of the tubing set is coupled to the container. There is a waste system that couples with at least one flexible tube member of the tubing set, and at least one flexible tube member is such that it carries sheath fluid to the flow nozzle.

Another embodiment of the invention is directed to a system for providing sterile sheath fluid and sample fluid to an analytic device. The system includes a sheath fluid delivery system and a sample fluid delivery system. The sheath fluid delivery system includes a tubing system comprising a plurality of flexible tube members, the tubing system for coupling with a closed container of sheath fluid, an analytic device and a waste system. The couplings define a pathway for sheath fluid from the closed container to the analytic device, the pathway being closed to the ambient environment. The system also includes a driver configured for placing pressure on a closed container of sheath fluid to move the sheath fluid through the tubing system. The sample fluid delivery system includes a pressurizable chamber for holding a closed container of sample fluid, the chamber receives pressurized fluid for driving the sample fluid from the closed container to the analytic device. There is also a fluid transport line for receiving sample fluid from its container in the chamber, the fluid transport line, when connected to a conduit in the chamber that connects to the closed container of sample fluid, defines a pathway for sample fluid from the closed container to the analytic device, that is closed to the ambient environment. The pressurizable chamber is oriented at an incline to facilitate sample fluid flow into the fluid transport line.

Another embodiment is directed to a system for providing sterile sheath fluid to an analytic device, for example, a flow cytometer. The system includes a tubing system comprising a plurality of flexible tube members, the tubing system configured for coupling with a closed container of sheath fluid, e.g., a flexible container, an analytic device and a waste system. The coupling defines a pathway for sheath fluid from the closed container to the analytic device, and the pathway is closed to the ambient environment. The system also includes a driver for placing pressure on the closed container of sheath fluid to move the sheath fluid through the tubing system.

Another embodiment of the invention is directed to an apparatus for sample fluid maintenance and transport. The apparatus includes a pressurizable chamber for receiving a container of sample fluid, an inlet for receiving pressurized gas into the pressurizable chamber, an outlet for sample fluid to exit the chamber, with the inlet being at an elevation above the outlet.

Another embodiment of the invention is directed to a method for transporting sheath fluid to a flow nozzle of a flow cytometer. The method includes providing a closed container of sheath fluid (for example, of a flexible material), providing a tubing set that allows for a sterile environment for the sheath fluid. The tubing set includes a plurality of flexible tube members, with at least one of the tube members of the plurality of tube members for coupling with each of a closed container of sheath fluid, a flow nozzle, and a waste system. A pathway for the sheath fluid is created that is closed to the ambient environment by coupling the closed container of sheath fluid to at least one flexible tube member and coupling at least one flexible tube member to the flow nozzle. The closed container is pressurized to drive the sheath fluid from the closed container to the flow nozzle.

Another embodiment of the invention is directed to a method for transporting sample fluid to an analytic device. The method includes providing an apparatus for sample fluid, the apparatus includes a pressurizable chamber for receiving a container of sample fluid, an inlet for receiving pressurized gas into the pressurizable chamber, an outlet for sample fluid to exit the chamber, with the inlet being at an elevation above the outlet. At least one closed container of sample fluid is then placed into the chamber, the closed container may be of a flexible material. At least one conduit for carrying sample fluid into the apparatus to the analytic device is then connected to the apparatus and the analytic device. The connection creates a pathway for the sample fluid from the apparatus to the analytic device that is closed to the ambient environment. The chamber is pressurized to drive the sample fluid from the container.

BRIEF DESCRIPTION OF THE DRAWINGS

Attention is now directed to the drawings, where like numerals and characters indicate like or corresponding components. In the drawings:

FIG. 1 is a diagram of a system of the contemporary art;

FIGS. 2A and 2B are diagrams of the flow nozzle detailing charging of the LAD;

FIG. 3 is a schematic diagram of the system of the invention with the sheath delivery system in accordance with an embodiment of the invention shown in detail;

FIG. 4 is a schematic diagram of an alternate embodiment of waste system connections in accordance with the invention;

FIG. 5 is a schematic diagram of the system with an alternate embodiment of the sheath delivery system;

FIG. 6 is a front perspective view of a sample fluid holding and delivering apparatus in accordance with an embodiment of the invention, with the cover removed;

FIG. 7is an exploded view of the apparatus of FIG. 6;

FIG. 8 is a top view of the apparatus of FIG. 6;

FIG. 9 is cross sectional view taken along line 9-9 of FIG. 8 of the apparatus;

FIG. 10 is top view of the apparatus of FIG. 6 with the cover removed; and,

FIGS. 11A and 11B are diagrams detailing the results of Examples 1 and 2 respectively.

DETAILED DESCRIPTION OF THE DRAWINGS

The present invention relates to a system for transporting sheath (also known as sheath fluid) and sample fluid from their respective sources to a flow nozzle of a flow cytometer. The system includes a plurality of tubes and connectors (junctions) for delivering both the sheath fluid and the sample fluid, as well as specialized holding devices, with driving mechanisms for both the sheath fluid and the sample fluid. Importantly, the system maintains a sterile environment. Specifically, the invention relates to a system for delivering sheath. Separately, a system and apparatus for delivering samples is contemplated. The systems can be used together and as part of a method. Throughout this document there may be references to directions such as up, down, upward, downward, above, below, etc. These directions are only exemplary, to allow for a description of the invention in a typical orientation.

FIG. 3 shows the system 15 of the invention in an exemplary operation, a Flow Activated Cell Sorting (FACS) operation on a flow cytometer 16. The flow cytometer 16 receives sheath fluid from a sheath fluid delivery system 20 and sample fluid from a sample fluid delivery apparatus 200. The sample fluid is delivered to the flow cytometer 16 via a delivery tube or line 242. The flow cytometer can be, for example, a MoFlo® sorting cytometer, or any other standard cytometer.

The system 20 of the invention, is shown in an exemplary operation. The system 20 includes a sheath fluid source, for example, a pressurized tank 21, containing sheath fluid, in a bag 22. The sheath fluid is, for example, sterile, pharmaceutical grade sheath fluid. A tubing system 24 (also known as a tubing set), connects this pressurized tank 21 to the flow nozzle 25 of a flow cytometer 16. While the pressurized tank has been demonstrated as effective when used with the present system, substitute devices may be used, as long as fluid sterility is maintained. Also, head pressure should be minimized. It is preferred for the sheath fluid to be placed in a bag that is of a sterile nature, can be squeezed to force fluid into the tubing system, and can be connected to the tubing system.

The tank 21 is typically of stainless steel or the like, and arranged so as to be sealed to retain pressure. The tank 21 is typically configured to withstand pressures of, for example, at least approximately 120 psi. The pressure will cause the sheath fluid to flow into the tubing system 24 (into the main line 28) from the bag 22, as pressure from the incoming pressurized gas will squeeze the bag 22, that is typically of a flexible material, forcing fluid out from it, or creating “bladder-type” action on the bag 22. The tank 21 is, for example, of an interior volume of approximately 3 gallons, and typically accommodates a container or bag 22 of sterile sheath fluid of volumes ranging, for example, from approximately 1 Liter to approximately 8 Liters, within its interior 27. The tank 21 typically sits in a sideways orientation, such that the container or bag 22 rests on a floor 27a (the floor 27a, formed for example, by a flat Plexiglas, polymeric, or other board-like insert) in the tank 21. The tank 21 is oriented, for example, such that its longitudinal axis LA is typically parallel to the horizontal or ground surface. A main line 28 of the tubing system 24 attaches to the tank 21.

A sealable cover 30 attaches to the tank 21, typically in a clamping-type or other suitable engagement, by a clamp 30a or the like. The cover 30 has a port 31 for the incoming pressurized gas, for example nitrogen, from a gas source (not shown), for example, a cylinder or compressed air from a mechanical compressor. This port 31 is, for example, formed of a female type quick connect, enabling the gas line that will attach thereat, to be of a corresponding male-type quick connect, for ease in operation. The cover 30 also includes ports for a relief valve 32, a pressure gauge 33, as well as an outflow port 34.

The outflow port 34 is typically formed of a first female quick connect 36, for extending into the interior 27 of the tank 21, and a second female quick connect 38, protruding from the cover 30. These female quick connects 36, 38 define a pathway for sheath fluid, from the bag 22 to the main line 28. The first female quick connect 36 joins with a male quick connect 42, that is on the end of a line 44 that extends from the bag 22. The second female quick connect 38 receives a male quick connect 45 of the main line 28.

The tubing system 24 provides a sterile environment, closed to the ambient environment, for the transport of sheath fluid from the bag 22 (source of sheath fluid) to the flow cytometer 16. The tubing system is comprised of a plurality of tubes, which are sealed to the ambient environment, can be sterilized, allows for the passage of fluid under 120 psi, and is generally of a flexible construction. The flow cytometer 16 can be, for example, a MoFlo® sorting cytometer or any other standard cytometer.

The main line 28 extends from the tank 21, specifically, the outflow port 34 to a Y-connector 50. The main line 28 is formed of tubing. A filter 54, for example, a single use filter, can be attached to, or integral with the main line 28. The attachment of the filter 54 to the main line 28 is typically through molded junctions 55, that are formed with the tubing set 24. The molded junctions 55 are sterile, and as such, maintain a sterile environment. The filter 54 can be of a variety of sizes, including a 0.22 micron filter or any other suitably sized filter for removing particulates. The particulates that are removed include salt.

The Y-connector 50 is such that it minimizes or eliminates the retention of air that may collect in the lines and sub lines, as detailed herein. This is because there is no dead space in the Y-connector 50 and no crevices or other interruptions or imperfections on the internal surface of the Y-connector 50. Also, the Y-connector 50 can be made of silicon, and as such has a smooth internal surface. These attributes of the Y-connector 50 allow for a more dynamic and less turbulent flow of the sheath fluid through the tubing system 24. Moreover, the Y-connector 50 does not affect the pressure in the tubing system 24.

The main line 34, at the Y-connector 50, divides into two sub lines 58, 59, also formed of tubing. The sub lines 58, 59 formed of tubing define a first flow stream (line 58) and a second flow stream (line 59). The sub lines 58, 59 can be fitted into pinch valves 60, 61, that are part of a table mounted bracket 64. The pinch valves 60, 61 can be actuated, to pinch the tubing of the sub lines 58, 59, such that fluid flow through the respective sub lines 58, 59 can be stopped at any time. Any of a variety of devices, however, can be used to occlude the inner lumens of the tubes that form the sub lines 58, 59, to stop fluid flow therethrough.

Each sub line 58, 59 terminates in a Y-connector 62, 63, that divides each sub line 58, 59 into two sets of sub lines 66, 68 and 71, 73, respectively. These sub lines 66, 68, 71, 73 are formed of tubing, as detailed further below. The Y-connectors 62, 63 are also formed in accordance with the Y-connector 50.

One sub line 66, 71 from each of the flow streams is coupled to a vacuum source (not shown), as part of a waste system (broken line block 74). The waste system 74 is typically preconfigured to receive the requisite sub lines 66, 71. The sub lines 66, 71 terminate at compression fittings 76, 77, with plugs 76a, 77a, at their ends. The compression fittings 76, 77, coupled with the plugs 76a, 77a, are the end of the “clean” or sterile portion of the system 20.

The compression fittings 76, 77, and their plugs 76a, 77a, are received in correspondingly shaped ports on check valves 78, 79. These check valves 78, 79 are one-way valves, biased so that material flowing through the sub lines 66, 71 can only flow unidirectionally, into the waste system 74, such that it does not backflow into the “clean” or sterile portion of the system 20. The check valves 78, 79 (and their ports for receiving the compression fittings 76, 77) are typically positioned in the table mounted bracket 64. The check valves 78, 79, by being mounted, must be cleaned by conventional sanitation methods. As a result, the check valves 78, 79 are not certifiably clean, and therefore are “dirty”. Thus, the check valves 78, 79 are the beginning of the “dirty” or nonsterile portion of the system 20.

In the “dirty” or non-sterile portion of the system 20, vacuum lines 82, 83 extend from the respective check valves 78, 79, and receive the flow of material from the respective sub lines 66, 71. The vacuum lines 82, 83 extend through valves 84, 85, that are for example, automatically actuated needle valves (but can also be manually actuated), that close the lumens of the vacuum lines 82, 83, respectively. These lines 82, 83 terminate at compression fittings 86, 87, that attach to vacuum sources (not shown).

Alternately, as shown in FIG. 4, sub lines 66 and 71 could include one-way check valves 78a, 79a, with connectors 78a′, 79a′ at their ends. These check valves 78a, 79a would be on the “clean” side of the system, with the “clean” side ending at the connectors 78a′, 79a′. The connectors 78a′, 79a′ would in turn, attach to the respective vacuum lines 82, 83.

The other sub lines 68, 73, extend from the respective Y-connectors 62, 63 to connector members 92, 93. These connector members 92, 93 are for example, molded tube junctions, and include reducer members, for reducing the fluid flow into constrictor tubes 94, 95 of smaller diameter, but at a flow of constant pressure, sufficient for introduction to the flow cytometer 16. The connector members 92, 93 define an end of the tubing set 24.

The constrictor tubes 94, 95 connect to the connector members 92, 93, and terminate at compression fittings 96, 97, with plugs 96a, 97a, at their ends. The compression fittings 96, 97 are configured for receipt by corresponding connector members of the flow cell nozzle 25 of the flow cytometer 16. This arrangement allows the tubing set 24 to couple with the flow nozzle 25 of the flow cytometer 16.

Because air is absent in the lines of the tubing set 24, and coupled with the release of sheath fluid from the tank 21 at a constant elevation, hydrostatic pressure at the nozzle 25 remains constant. This constant pressure allows for stability in aerosol formation for the process of FACS, as detailed in Example 1 below.

The tubing for the lines 28, 58, 59, 66, 68, 71 and 73 can be of a polymeric material, for example, silicon, that is clear, and at least translucent, and may be transparent, allowing the user to see the fluid flow inside the lines. The tubing is also autoclavable. It is typically of a small internal diameter, for example, approximately {fraction (1/16)} inch, when compared to the internal thickness, for example, with an outer diameter of approximately {fraction (5/16)} inches, as it is designed to accommodate fluid moving through it at high pressures, typically up to approximately 120 psi. Other sizes and dimensions may be used, so long as the tubing does not rupture. Additionally, a variety of materials may be used, as long as the material can be sterilized and allows for passage of the fluid under sufficient pressure.

The Y-connectors 50, 62 and 63 are positioned between the lines 66, 68, 71, 73, to facilitate bi-directional fluid flow through the flow nozzle 25. This positioning makes it possible to remove air bubbles or other items in the system that might disrupt laminar flow and hydrodynamic focusing.

The Y-connectors 50, 62 and 63 are, for example, one-piece molded plastic, polymeric or elastomeric members that are autoclavable. They are typically of the same internal diameter as the aforementioned tubing (for the lines and sublines), so as to form a smooth fit with the tubing that connects thereto, free of any bumps, edges or ridges, that could cause turbulence in the tubing system (set) 24 or bubbles to form therein. They are typically clear, transparent, or translucent, allowing the user to view the fluid flow therein.

The tubing set 24 can be assembled, sterilized and packaged by a manufacturer. As a result, the tubing set 24 can be removed from the sterile packaging upon its use, which is typically a single or one-time use.

FIG. 5 is directed to an alternate sheath fluid delivery system 120. This system 120 is a completely “clean” system, as waste is deposited directly into a tank 121, such that the waste can not reenter the system 120, and all components of the tank 121 and tubing set 24 are autoclavable. By sending waste directly to a sealed waste bag under suction pressure, the waste fluid contacts the inner lumen of the tank connector and enters into a closed bag under suction. Accordingly, the system 120 lacks a “dirty” side, like the system 20 detailed above. Components of this system 120 are similar to components of the system 20, detailed above, except where indicated. Accordingly, similar components, that have been described above, are shown in FIG. 5, but not described.

In this system 120, the sub-lines 66, 71 extend from their respective Y-connectors 62, 63, toward the waste tank 121. The waste tank 121 includes an inner chamber 129 for receiving waste and is constructed to allow for proper disposal of biological waste. This includes autoclaving the entire tank 121. The sub lines 66, 71 terminate in quick connects 132, 133, that are, for example, male quick connects. These quick connects 132, 133 attach to corresponding quick connects 134, 135, that are, for example, female quick connects. The quick connects 134, 135 are typically attached to check valves 136, 137, that open only in the direction (for example, downward) of the waste tank 121.

The check valves 136, 137 terminate in threaded fittings 142, 143, that attach to corresponding fittings 144, 145 on the cover 146 of the tank 121. The cover 146 also includes ports 150, 151 to which a vacuum (suction) (not shown) is connected. The vacuum, by providing a suction force, draws waste from the sub lines 66, 71, into the tank 121, when the respective pinch valves 154, 155 are open.

Pinch valves 154, 155 are typically along the sub lines 66, 71. These pinch valves 154, 155 function similarly to the pinch valves 60, 61, detailed above, and also sit in the table mounted bracket 64.

Turning now to FIGS. 6-10, an apparatus 200 may be used with the system. Also, use of the system includes methods for storing and transporting sample fluid to a flow nozzle 25 of a flow cytometer 16 (FIGS. 3 and 5). Once the sample fluid reaches the nozzle 25, the sample fluid combines with the sheath fluid for sorting and analysis.

FIGS. 6-10 show the sample fluid delivery apparatus 200 of the invention. The apparatus 200 includes a pressurizable unit 202 for holding a container 204 of sample fluid, in a chamber 205 in the interior of the unit 202. The container 204 is, for example, a flexible container, such as an intravenous bag of sample fluid, holding sample cells or single cell suspensions, with volumes ranging from approximately 5 ml to approximately 100 ml. The unit 202 is typically inclined on an angled support 206, that rests on a rocking platform 208. The rocking platform 208 is controlled by a motor (not shown) and associated driving apparatus (not shown), housed within body 210. The body 210, also includes, for example, an ON/OFF switch 212 and rocker control 213.

The unit 202 is typically separable from the support 206. Accordingly, it can be taken off of the support 206, for example, when installation, positioning, or removal of a container 204 is desired.

The unit 202 is formed by a base 214, for example, a cylindrical structure. The base 214 includes a body or cylindrical portion 216, with an outwardly extending platform 218. The base is typically a one-piece unitary member formed of metal that is thermally conductive, or other suitable thermally conductive material.

A cover 220 encloses the unit 202. This cover 220 typically extends to the edge of the platform 218. The cover 220 is typically made of metal, such as that for the base 214. The cover 220 could also be of a transparent or translucent material, or be of metal with one or more transparent or translucent portions.

A gasket 222 fits between the platform 218 of the base 214 and the cover 220. A clamp 224, extends around the periphery of the platform 218 and cover 220.

The base 214 includes a floor 230 on which the container 204 of sample fluid, here, for example, a bag of sample fluid, is held. The container 204 can be held in place by pins, hooks, or the like, that fit into opening in the floor 230 and extend into the opening 204a in the container 204, i.e., the bag.

The base 214 includes an opening 240, that receives a bulkhead fitting 241. The line 242, for example, that is formed of tubing, connects to this bulkhead fitting 241 (a sterile through-hole). This connection allows sample fluid from the container 204 to reach the line 242, for transport to the flow cytometer 16.

The bulkhead fitting 241 includes a tube portion 241a for receiving a neck 204b of the container 204. The bulkhead fitting 241 has collar members 241b, 241c at a first end 241d (to remain inside of the base 214) and an external nut 241e that fits over a threaded nose 241f of the tube portion 241a, at the end 241g of the bulkhead fitting 241, that extends outside of the base 214, through the opening 240. The external nut 241e secures the bulkhead fitting 241 in place. The bulkhead fitting 241 fits within the opening 240 to form an air-tight (fluid-tight) seal. It is preferred that the flow of sample fluid into this line 242 be downward (with gravity), to eliminate or greatly inhibit air bubbles from forming in the sample fluid. If air bubbles exist, they will rise, and therefore, not introduce air into the flow nozzle. This is because while air bubbles may be present in the sample fluid, they can not be present in the flow nozzle. The bulkhead fitting 241, at its end 241f, connects to the line 242, through which sample fluid is transported to the flow nozzle (not shown) of the flow cytometer (not shown). A pinch valve (not shown) may be placed along the sample tubing 242, if desired.

An opening 250 in the base 214, opposite the opening 240, receives a pressure gauge 254 at its stem 255. The pressure gauge 254 is typically a dial gauge, allowing for a visual indication of the pressure in the chamber 205. The stem 255 includes a fitting 256 at its end that when in the opening 250 forms an air-tight (fluid-tight) seal. Arms 258, 259 extend from the pressure gauge 254. One arm 258 receives an air line 260 that supplies pressurized air to the chamber 205. This air line 260 typically connects to a source of pressurized air 261 for pressurizing the chamber 205 and forcing fluid from the container 204, by creating a bladder effect on the container 204.

The opposite arm 259 receives a pressure relief valve (V) 262. The pressure relief valve 262 is typically preset to open when a designated (predetermined) pressure is reached in the chamber 205, for example, approximately 120 psi. Alternately, the pressure relief valve 262 can be manually or automatically activatable. The pressure relief valve 262 can be a ball valve (preset by a spring loading to a desired pressure) with a manual override or the like. This valve 262 can also be such that it is manually set by a thumb screw mechanism or the like.

Turning specifically to FIG. 10, within the floor 230 is a channel 265 for coolant circulation, for cooling the floor 230 of the base 214 and the chamber 205. The channel 265 includes a main channel 266, that is, for example U-shaped, that allows for fluid circulation. An auxiliary channel 268 joins to this main channel 266 from an edge of the base 214. The opening 268a of this auxiliary channel 268 is typically closed with a plug or the like.

Coolant, typically fluid, liquid or gaseous (from a source 269), and, for example, water, is supplied to the channel 265 in the base 214 through a line 270, that connects to the channel 265 at a port 271 (including an opening 271a in the base 214). Coolant exits the channel 265 through a port 272 (including an opening 272a in the base 214), that connects to a line 273 for outflow. Once in the outflow line 273, the coolant can be recycled or exited from the apparatus 200. The lines 270, 273, are typically tubes, that connect to the respective ports 271, 272 through fittings 276, 277. The channel 265 is not in contact with the chamber 205, so coolant does not enter the chamber 205.

The clamp 224 is typically C-shaped (with a U-shaped inner groove 278) and extends around a substantial portion of the periphery of the platform 218 and cover 220. Coupled with the gasket 222, the clamp 224 seals the chamber 205, making it air and water tight. The chamber 205, when sealed, typically accommodates pressures up to approximately 120 psi.

The clamp 224 is formed of peripheral members 279, 280 that pivot about a joint 282. The ends of the peripheral members 279a, 280a, include protrusions 279b, 280b with openings 279c, 280c that accommodate a threaded pin 284 and adjustable bolt 286, in order that the clamp 224 be adjusted to the desired tightness.

With the clamp 224 removed, the cover 220 can be lifted off of the gasket 222. This allows access to the chamber 205, typically for installing, positioning or removing bags of sample fluid in the chamber 205.

The angled support 206 is typically at an angle θ (FIG. 9), which permits the opening 240 for sample fluid leaving the apparatus 200 to be at a lower elevation than the opening 250 for pressurized gas of the base 214. For example, the angle θ can be approximately 15 degrees to approximately 35 degrees, and typically, is approximately 25 degrees. This allows for sterility of the sample fluid and the absence of air bubbles in the line 242, through which it travels to the flow nozzle 25 (of the flow cytometer 16). The platform 208 on which the angled support 206 sits, is rocked at various intervals, to prevent the sample fluid from settling.

The unit 202 typically sits on the angled support 206, such that its axis 290 (FIG. 8) is parallel to the axis 292 (FIG. 7) of the support 206. The angled support 206 typically sits on the platform 208, such that it is rocked from side to side. As a result, the container 204 rocks in the unit 202 from side to side. Alternately, the positions of the unit 202 with respect to the angled support 206, and the angled support 206 with respect to the platform 208 may be changed, such that the container 204 rocks up and down (in the direction of the incline of the angled support 206).

EXAMPLES

Example 1

A sheath tank, a stainless steel cylindrical tank with an approximately 8 inch diameter, a depth of at least 13⅜ inches, and able to withstand pressures of up to 100 psi when capped, was modified with an internal cylindrical tube (straw) that extended approximately 13 inches into the tank, to a point just above the floor. The tank was filled with approximately 8 liters of Dulbecco's Phosphate Buffered Saline (PBS). The cap had connections for pressure in, fluid out, and a pressure gauge. The pressure gauge included a differential pressure measuring (DPM) unit, that attached between the pressure in and fluid out connections. The differential pressure gauge measured the difference between gas pressure at the top of the tank and pressure of the fluid as it leaves the tank. The resultant readings are shown in FIG. 11A in a diagram of differential pressure (in pounds per square inch (psi) versus time (in minutes)).

The tank was sealed with the cap, so as to be air and water tight. A sterile sheath delivery tubing system (tubing set) in accordance with the tubing system 24 detailed above, was attached to the tank cap at the fluid out port and its differential pressure measuring (DPM) unit, via a quick connect. The tubing set was placed into a table mounted bracket, as detailed above. Other ends of the tubing set were attached to a flow nozzle of a flow cytometer and a waste tank, respectively.

The sheath tank was pressurized to approximately 60 psi using a cylinder of compressed Nitrogen gas. The pressurized Nitrogen gas drove the fluid from the tank, through the straw, and into the tubing system, to the flow nozzle. Fluid exited the flow nozzle through a 70 micrometer tip. The flow nozzle was inverted to force and ensure that all trapped air was out of the nozzle. Upon observation, no air was trapped in the tubing set and the flow nozzle. The nozzle was installed in its proper orientation on the flow cytometer and a drop drive frequency of approximately 94,000 Hz was empirically selected. This frequency was optimal for the creation of a last attached drop (LAD), in a fluid stream nearest to the flow nozzle. This frequency was confirmed by visual observation on a monitor, associated with the flow cytometer. The camera that visualized the LAD was subjected to a stroboscope and the images were captured using a MATROX RTX 100 video capture card and Adobe® Premiere Pro software, on a personal computer (PC). Once the LAD was established, the system pressure, drop drive frequency, drop drive amplitude, drop drive phase, stroboscope phase and camera position were kept constant and not altered during the course of the sheath fluid flow from the sheath tank to the flow nozzle, known hereafter as “the run”. With the LAD established, the run began as a time zero (to) image captured at zero minutes (t₀=0 minutes). DPM measurements were taken at 5 second intervals during the run.

The run was recorded and lasted for an approximately 11 hour period. Measurements were taken of the pressure at to, which was at 0 minutes and a later time (t₁), which was at 662 minutes, near the end of the run, these measurements shown in FIG. 11A. Also, at times t₀ and t₁, images (snapshots) of the respective sheath fluid streams, with their LAD, were taken and are shown as superimposed images in the diagram of FIG. 1A. The image taken at time t₁ is also known as the “end of run” image.

After approximately 11 hours, the run (sheath fluid flow) was halted and the DPM was deactivated. A differential pressure change of 0.31 psi to 0.48 psi coincided with a distance change of the LAD, moving toward the flow nozzle (upward) approximately 280 micrometers.

Based on pressure readings and visual observations of the LAD for the fluid stream from the flow nozzle, a differential pressure change coincided with the distance of the LAD moving toward the flow nozzle as time increased.

This corroborates the situation described above and as shown in FIG. 2B.

Example 2

The tank of Example 1 was used, with the internal cylindrical tube removed, and a 5 liter bag of Dulbecco's PBS was placed into the interior of the tank. A Plexiglas board was also placed into the tank. The bag, at a line extending from the bag, was attached to the fluid out port, this fluid out port being the port that formerly accommodated the internal cylindrical tube (straw). This port also included a quick connect on the outside of the tank cover. The cap was sealed on the body of the tank, such that the tank was air and water-tight.

The tank was connected to the tubing set 24 as detailed above for Example 1. The tank was tilted to a horizontal position, where the bag rested on the Plexiglas board and the line that extended from the bag was also supported by this Plexiglas board. The tank was oriented so that the fluid out port, that accommodated the line that extended from the bag, was at a six-o'clock position. The tank was pressurized to approximately 60 psi using a cylinder of compressed Nitrogen gas. The pressurized Nitrogen gas drove the fluid from bag, through the port and into the tubing system, to the flow nozzle. Fluid exited the flow nozzle through a 70 micrometer tip. The flow nozzle was inverted to force and ensure that all trapped air was out of the nozzle. Upon observation, no air was trapped in the tubing set and the flow nozzle.

The tank was pressurized to 60 psi. The nozzle was installed in its proper orientation on the flow cytometer and a drop drive frequency of approximately 94,000 Hz was empirically selected. This frequency was optimal for the creation of a LAD, in a fluid stream nearest to the flow nozzle. This frequency was confirmed by visual observation on a monitor, associated with the flow cytometer. The camera that visualized the LAD was subjected to a stroboscope and the images were captured using a MATROX RTX 100 video capture card and Adobe® Premiere Pro software, on a personal computer (PC). Once the LAD was established, the system pressure, drop drive frequency, drop drive amplitude, drop drive phase, stroboscope phase and camera position were kept constant and not altered during the course of the sheath fluid flow from the sheath tank to the flow nozzle, known hereafter as “the run”. With the LAD established, the run began as a time one (t₁′) image was captured at 31 minutes (t₁′=31 minutes). DPM measurements were taken at 5 second intervals during the run, from time zero (t₀′=0 minutes).

The run was recorded and lasted for an approximately 10 hour period. Measurements of the pressure were recorded at times t₁′, which was 31 minutes and t₂′, which was at 592 minutes, as shown in FIG. 11B. Also, at times t₁′ and t₂′, images (snapshots) of the respective sheath fluid streams, with their LAD, were taken at times t₁′ and t₂′ (the image at t₂′ also being known as the “end of run” image), and are shown as superimposed images in the diagram of FIG. 11B.

After approximately 10 hours, the run (sheath fluid flow) was halted and the DPM was deactivated. After approximately 10 hours, the LAD position remained constant, with respect to the distance from the flow nozzle. The differential pressure remained constant at −0.09 psi, until the run was halted at a time t₃′, which was 607 minutes.

Based on pressure readings and visual observations of the LAD for the fluid stream from the flow nozzle, the lack of differential pressure change over the 10 hour time period coincided with the distance of the LAD with respect to the flow nozzle remaining constant, as time increased.

This corroborates the situation described above and as shown in FIG. 2A.

While the present invention is typically used with flow cytometers for sorting cells, it is also useful with flow cytometers for sorting other particles, typically similar in size to cells. These particles include chromosomes, cellular organelles or non-living particles such as beads.

There have been shown and described preferred embodiments of sample fluid and sheath fluid apparatus and delivery systems. It is apparent to those skilled in the art, however, that many changes, variations, modifications, and other uses and applications for the systems and their components are possible, and also such changes, variations, modifications, and other uses and applications which do not depart from the spirit and scope of the invention are deemed to be covered by the invention, which is limited only by the claims which follow.

Claims

1. A molded tubing set for coupling a source of sheath fluid and a flow nozzle, the tubing set comprising:

(a) at least one flexible tube member, which can transport fluid at pressures of approximately 120 psi, the tube member configured for providing a sterile environment for fluid transport from the sheath fluid source to the flow nozzle; and

(b) a connection to the source of sheath fluid, whereby the connection forms a sealed attachment that prevents exposure to ambient environment.

2. The tubing set of claim 1, the connection between the tube member and the source is fitted and removable.

3. The tubing set of claim 1 comprising plurality of flexible tube members, at least one Y-connector, and a waste system.

4. The tubing set of claim 1, comprising: a first flexible tube member in communication with a first Y-connector, at least two second flexible tube members in communication with the first Y-connector and each of the at least two second flexible tube members in communication with respective second Y-connectors, and at least two third flexible tube members in communication with each of the second Y-connectors.

5. The tubing set of claim 1 comprising at least one connector fittings for attachment to the flow nozzle.

6. The tubing set of claim 3 comprising: compression fittings for connecting the tubing set to the waste system.

7. A method for transporting sheath fluid to a flow nozzle of a flow cytometer comprising:

(a) affixing a sterile bag of sheath fluid to a tubing set, whereby a sterile fitted connection is formed, the tubing set comprising at least one flexible tube member;

(b) attaching at least one flexible tube member to the flow nozzle;

(c) applying pressure to the bag of sheath fluid to cause the sheath to flow from the bag to the tube member.

8. The method of claim 7, comprising applying pressure to the bag by placing the bag in a tank, with the tank in communication with a source of pressurized gas, the tank includes a port where the closed container is coupled with at least one flexible tube member, and provides a longitudinal axis extending lengthwise through the tank.

9. The method of claim 8, wherein the tank is such that its longitudinal axis is at least substantially parallel to the horizontal.

10. The method of claim 9, wherein the tank is oriented such that the second port is below the first port.

11. The method of claim 1 comprising attaching flexible tube members to a waste system, whereby compression fittings are used to facilitate attachment.

12. The method of claim 11, wherein the waste system includes a waste tank.

13. A system for transporting sheath fluid to a flow nozzle of a flow cytometer, comprising:

(a) a flexible container of sheath fluid;

(b) a tubing set for coupling with the container of sheath fluid, a waste system, and the flow nozzle, the tubing set comprising: a plurality of flexible tube members which can transport fluid at pressures of approximately 120 psi and provides a sterile environment for sheath fluid transport from the container to the flow nozzle;

(c) a pressurizable tank for holding the container of sheath fluid, the tank including at least a first port for receiving pressurized gas for driving the fluid in the container through the tubing set, and, a second port where at least one of the plurality of flexible tube members of the tubing set is in communication with the container; and,

(d) a waste system in communication with at least one flexible tube member of the tubing set.

14. The system of claim 13, wherein the tubing set, in communication with the container and the flow nozzle, defines a pathway for sheath fluid that is closed to the ambient environment.

15. The system of claim 13, comprising a first flexible tube member in communication with a first Y-connector, at least two second flexible tube members in communication with the first Y-connector and each of the at least two second flexible tube members in communication with respective second Y-connectors, and at least two third flexible tube members in communication with each of the second Y-connectors.

16. The system of claim 13, comprising a source of pressurized gas in communication with the tank.

17. The system of claim 13, wherein the waste system defines a non-sterile side of the system.

18. The system of claim 17, wherein the waste system includes at least one waste line for coupling with at least one flexible tube member of the tubing set, the at least one waste line in communication with a source of suction.

19. The system of claim 13, wherein the waste system includes a waste tank.

20. The system of claim 19, comprising: at least one one-way valve in communication with the waste tank for receiving the at least one flexible tube member of the tubing set, the at least one way valve biased to prevent fluid backflow into the at least one flexible tube member.

21. The system of claim 13 comprising a plurality of Y-connectors attached to the tubing set.

22. A system for providing sterile sheath fluid and sample fluid to an analytic device, comprising:

a sheath fluid delivery system including: at least one flexible tube member, configured for coupling with a closed container of sheath fluid and the analytic to define a pathway for sheath fluid from the closed container to the analytic device, the pathway being closed to the ambient environment; and,

a sample fluid delivery system including: a fluid transport line in communication with the container defining a pathway for sample fluid from the closed container to the analytic device that is closed to the ambient environment.

23. The system of claim 22, wherein the sheath fluid delivery system includes a first tank configured for being pressurized and holding a closed container of sheath fluid.

24. The system of claim 23, wherein the first tank includes at least one port configured for communication with a corresponding portion of the tubing system for facilitating sheath fluid transport through the tubing system.

25. The system of claim 22, wherein the sheath fluid delivery system includes a waste system for receiving waste, the second tank configured for coupling with a flexible tube member, and a one-way valve system.

26. The system of claim 22, wherein the closed container of sample is placed in a pressurizable chamber that includes an inlet for pressurized gas and an outlet for sample fluid.

27. The system of claim 22, wherein the sample fluid delivery system includes a rocking mechanism in communication with the pressurizable chamber.

28. The system of claim 26, wherein the pressurizable chamber includes a floor configured for accommodating coolant circulation therethrough.

29. A system for providing sterile sheath fluid to an analytic device comprising:

(a) a closed container of sheath fluid;

(b) a tubing system comprising a plurality of flexible tube members, the tubing system configured for coupling with the container, the analytic device, and a waste system, to define a pathway for sheath fluid from the closed container to the analytic device, the pathway being closed to the ambient environment; and,

(c) a driver configured for placing pressure on the closed container of sheath fluid to move the sheath fluid through the tubing system.

30. The system of claim 29, comprising: a first tank configured for being pressurized and holding the closed container of sheath fluid.

31. The system of claim 29, wherein the sheath fluid delivery system includes a second tank for receiving waste, the second tank configured for coupling with a portion of the tubing system.

32. The system of claim 29, wherein the sheath fluid delivery system includes a waste system configured for coupling with the tubing system.

33. An apparatus for sample fluid maintenance and transport comprising:

(a) a removable bag which contains sample fluid;

(b) a pressurizable chamber for receiving the bag of sample fluid;

(c) an outlet for sample fluid to exit the chamber; and,

(d) an inlet, for receiving pressurized gas into the pressurizable chamber.

34. The apparatus of claim 33, wherein the chamber is inclined.

35. The apparatus of claim 33, wherein the sample fluid is sterile.

36. The apparatus of claim 33, comprising a source of pressurized gas in communication with the inlet of the chamber.

37. The apparatus of claim 35, comprising an angled support member for supporting the chamber on an incline.

38. The apparatus of claim 33, comprising a rocking mechanism in communication with the chamber.

39. The apparatus of claim 37, wherein the angled support member is positioned intermediate the chamber and the rocking mechanism.

40. The apparatus of claim 33, wherein the chamber includes a floor, the floor including a coolant circulation system therein.

41. The apparatus of claim 33, wherein the chamber includes a removable cover, for providing access to the interior of the chamber.

42. A method for transporting sample fluid to an analytic device comprising:

placing a sealed bag of sample in a pressurizable chamber;

connecting the sealed bag of sample to at least one conduit for carrying the sample fluid into communication with the analytic device to create a pathway for the sample fluid to the analytic device, that is closed to the ambient environment; and

pressurizing the chamber to drive the sample fluid.

43. The method of claim 42, comprising: rocking the chamber to inhibit settlement of the sample fluid.

44. The method of claim 42, wherein the pressurizing the chamber includes activating a gas source for supplying pressurized gas to the chamber.

45. A system for providing sterile sheath and sample fluid to an analytic device, comprising:

(a) a closed container of sheath fluid;

(b) a closed container of sample fluid;

(c) a plurality of flexible tube members and Y-connectors configured for coupling with the closed container of sheath fluid, the analytic device and a waste system;

(d) a driver configured for placing pressure on the closed container of sheath fluid;

(e) at least one, one-way valve for connecting the tube member to the waste system;

(f) a pressurizable chamber for holding the closed container of sample fluid; and

(g) a fluid transport line in communication with the sample fluid and the analytic device that is closed to the ambient environment.