U.S. patent application number 10/862268 was filed with the patent office on 2005-01-20 for fluid delivery system for a flow cytometer.
Invention is credited to Haug, Jeffrey S..
Application Number | 20050011582 10/862268 |
Document ID | / |
Family ID | 34069328 |
Filed Date | 2005-01-20 |
United States Patent
Application |
20050011582 |
Kind Code |
A1 |
Haug, Jeffrey S. |
January 20, 2005 |
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.
Inventors: |
Haug, Jeffrey S.; (Leawood,
KS) |
Correspondence
Address: |
POLSINELLI SHALTON WELTE SUELTHAUS P.C.
700 W. 47TH STREET
SUITE 1000
KANSAS CITY
MO
64112-1802
US
|
Family ID: |
34069328 |
Appl. No.: |
10/862268 |
Filed: |
June 7, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60476380 |
Jun 6, 2003 |
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60504105 |
Sep 19, 2003 |
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60526747 |
Dec 3, 2003 |
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Current U.S.
Class: |
141/65 |
Current CPC
Class: |
G01N 2015/1409 20130101;
G01N 15/1404 20130101; G01N 2015/149 20130101 |
Class at
Publication: |
141/065 |
International
Class: |
B65B 031/04 |
Claims
What is claimed is:
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.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] 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
[0002] 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
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] The sample fluid system lacks any in-line valves, and
includes sterile tubing that is removable, replaceable and
autoclavable.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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
[0030] Attention is now directed to the drawings, where like
numerals and characters indicate like or corresponding components.
In the drawings:
[0031] FIG. 1 is a diagram of a system of the contemporary art;
[0032] FIGS. 2A and 2B are diagrams of the flow nozzle detailing
charging of the LAD;
[0033] 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;
[0034] FIG. 4 is a schematic diagram of an alternate embodiment of
waste system connections in accordance with the invention;
[0035] FIG. 5 is a schematic diagram of the system with an
alternate embodiment of the sheath delivery system;
[0036] 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;
[0037] FIG. 7is an exploded view of the apparatus of FIG. 6;
[0038] FIG. 8 is a top view of the apparatus of FIG. 6;
[0039] FIG. 9 is cross sectional view taken along line 9-9 of FIG.
8 of the apparatus;
[0040] FIG. 10 is top view of the apparatus of FIG. 6 with the
cover removed; and,
[0041] FIGS. 11A and 11B are diagrams detailing the results of
Examples 1 and 2 respectively.
DETAILED DESCRIPTION OF THE DRAWINGS
[0042] 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.
[0043] 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.RTM. sorting cytometer, or
any other standard cytometer.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.RTM. sorting cytometer or any other standard cytometer.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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).
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] The angled support 206 is typically at an angle .theta.
(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 .theta.
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.
[0085] 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
[0086] A sheath tank, a stainless steel cylindrical tank with an
approximately 8 inch diameter, a depth of at least 133/8 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)).
[0087] 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.
[0088] 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.RTM. 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 (t.sub.0=0 minutes). DPM measurements were taken at 5
second intervals during the run.
[0089] 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 (t.sub.1), which was at 662 minutes,
near the end of the run, these measurements shown in FIG. 11A.
Also, at times t.sub.0 and t.sub.1, 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 t.sub.1 is also known as the "end of run" image.
[0090] 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.
[0091] 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.
[0092] This corroborates the situation described above and as shown
in FIG. 2B.
Example 2
[0093] 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.
[0094] 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.
[0095] 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.RTM. 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 (t.sub.1') image was
captured at 31 minutes (t.sub.1'=31 minutes). DPM measurements were
taken at 5 second intervals during the run, from time zero
(t.sub.0'=0 minutes).
[0096] The run was recorded and lasted for an approximately 10 hour
period. Measurements of the pressure were recorded at times
t.sub.1', which was 31 minutes and t.sub.2', which was at 592
minutes, as shown in FIG. 11B. Also, at times t.sub.1' and
t.sub.2', images (snapshots) of the respective sheath fluid
streams, with their LAD, were taken at times t.sub.1' and t.sub.2'
(the image at t.sub.2' also being known as the "end of run" image),
and are shown as superimposed images in the diagram of FIG.
11B.
[0097] 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 t.sub.3',
which was 607 minutes.
[0098] 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.
[0099] This corroborates the situation described above and as shown
in FIG. 2A.
[0100] 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.
[0101] 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.
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