U.S. patent application number 10/286282 was filed with the patent office on 2003-03-20 for method and apparatus for the concentration of fluid-borne pathogens.
Invention is credited to Brown, Richard I., Min, Kyungyoon.
Application Number | 20030054934 10/286282 |
Document ID | / |
Family ID | 25364433 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030054934 |
Kind Code |
A1 |
Brown, Richard I. ; et
al. |
March 20, 2003 |
Method and apparatus for the concentration of fluid-borne
pathogens
Abstract
Methods and apparatus for concentrating and recovering pathogens
from a fluid other than blood are disclosed. The method includes
concentrating the pathogens contained in the fluid by continuously
feeding the fluid through one or more flexible chamber(s) and
subjecting the chamber(s) to centrifugal forces. The concentrated
pathogens may be re-suspended by shaking the chamber(s).
Inventors: |
Brown, Richard I.;
(Northbrook, IL) ; Min, Kyungyoon; (Gurnee,
IL) |
Correspondence
Address: |
Bradford R. L. Price, Esq.
Baxter Healthcare Corporation
Fenwal Division - RLP 30, Route 120 & Wilson Road
P. O. Box 490
Round Lake
IL
60073
US
|
Family ID: |
25364433 |
Appl. No.: |
10/286282 |
Filed: |
November 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10286282 |
Nov 1, 2002 |
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09874731 |
Jun 5, 2001 |
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6500107 |
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Current U.S.
Class: |
494/21 ; 494/27;
494/45 |
Current CPC
Class: |
A61M 1/362 20140204;
A61M 2202/20 20130101; B04B 2005/045 20130101; A61M 1/3696
20140204; A61M 1/3693 20130101; C12M 33/10 20130101; B04B 5/0428
20130101; B04B 5/0442 20130101 |
Class at
Publication: |
494/21 ; 494/27;
494/45 |
International
Class: |
B04B 011/02 |
Claims
That which is claimed:
1. A method for concentrating and recovering pathogens from fluid,
comprising the steps of: continuously feeding the fluid through a
flexible centrifugation chamber, subjecting the centrifugation
chamber to centrifugal forces by rotating said chamber about a
rotational axis while fluid is being fed therethrough to
concentrate in said chamber pathogens contained in the fluid, and
shaking the chamber to re-suspend pathogens concentrated in the
chamber.
2. The method of claim 1 which includes twisting the chamber.
3. The method of claim 1 in which the step of shaking is carried
out manually.
4. The method of claim 1 in which the flexible centrifugation
chamber is elongated, and fluid is introduced into the chamber at
substantially one end thereof and withdrawn at substantially the
other end thereof.
5. The method of claim 1 in which the flexible centrifugation
chamber is elongated, and fluid is introduced into the chamber at
substantially one end thereof and withdrawn at substantially the
same end thereof after traversing the length of the container at
least about twice.
6. The method of claim 1 in which said chamber is elongated and
includes a transverse seal line dividing the chamber into at least
two sub-chambers, each sub-chamber including an inlet and an outlet
generally in proximity to said transverse seal line and a
segment-defining seal line separating said inlet and outlet and
defining first and second flow path segments for fluid passing
through said subchamber.
7. The method of claim 1 in which said chamber is defined by pair
of facing plastic sheets, each of which has a peripheral edge area,
said sheets being sealed together along at least said peripheral
area.
8. The method of claim 1 in which the fluid is water.
9. A method for concentrating and recovering pathogens from fluid,
comprising the steps of: continuously feeding the fluid serially
through a plurality of flexible centrifugation chambers, subjecting
the centrifugation chambers to centrifugal forces by rotating said
chambers about a rotational axis while fluid is being fed
therethrough to concentrate in said chamber pathogens contained in
the fluid, and shaking at least one of the chambers to re-suspend
pathogens concentrated in the chamber.
10. The method of claim 9 including adding a sedimentation
enhancing agent to the fluid after processing through a
chamber.
11. The method of claim 9 in which each of the one and other
containers includes a flow path through which the fluid is fed, and
the flow path in said other chamber is of greater length than the
flow path in said one chamber.
12. The method of claim 9 further comprising the step of sealing
and storing one of the chambers.
13. A method for concentrating and recovering pathogens from fluid,
comprising the steps of: continuously feeding the fluid through a
flexible centrifugation chamber, subjecting the centrifugation
chamber to centrifugal forces by rotating said chamber about a
rotational axis while fluid is being fed therethrough to
concentrate in said chamber pathogens contained in the fluid, and
shaking the chamber to and fro to cause a bolus of fluid in said
chamber to slosh back and forth, creating increased shear forces in
said chamber and causing said chamber to flex outwardly as said
bolus of fluid sloshes back and forth, to re-suspend pathogens
concentrated in the chamber.
14. The method of claim 13 including removing concentrated
particles from said chamber.
15. The method of claim 13 in which the fluid is thereafter fed
through another chamber to concentrate pathogens therewith.
16. A disposable fluid circuit for use in a centrifuge, said
circuit including a plurality of flexible plastic separation
chambers adapted for cooperation with a rotatable centrifuge
platform, said chambers being in parallel fluid flow communication
in said circuit.
17. A disposable fluid circuit for use in a centrifuge for
concentrating pathogens from fluid, said circuit including at least
a first and second flexible plastic separation chambers adapted for
cooperation with a rotatable centrifuge platform, said chambers
being in series fluid flow communication in said circuit, each of
said chambers having a flow path defined therein for fluid flow
through the chamber, said first chamber including a flow path for
removal of particles concentrated therein, and said second chamber
being adapted to receive processed fluid from the first chamber and
having a flow path therein to define a substantially uniform flow
field.
18. The fluid circuit of claim 17 in which each chamber comprises a
separate flexible plastic container.
19. The fluid circuit of claim 17 in which each chamber comprises a
sub-chamber of a single integral flexible plastic container.
20. A disposable fluid circuit for use in a centrifuge for
concentrating pathogens from fluid, said circuit including at least
a first and second flexible plastic separation chambers adapted for
cooperation with a rotatable centrifuge platform, said chambers
being in series fluid flow communication in said circuit, each of
said chambers having a flow path defined therein for fluid flow
through the chamber, said first chamber being adapted to carry out
a first separation process on said fluid and said second chamber
being adapted to receive processed fluid from the first chamber and
to concentrate pathogens contained in such processed fluid, said
fluid circuit including a source of a sedimentation enhancing agent
and an agent flow path communicating between said agent source and
an entry site in said fluid circuit for addition to said processed
fluid.
21. The disposable fluid circuit of claim 20 in which said entry
site is in a fluid flow path communicating between said first and
second chambers.
22. The disposable fluid circuit of claim 20 in which said entry
site is in said second chamber.
Description
[0001] The present invention relates to method and apparatus for
concentrating fluid-borne pathogens from fluids potentially
containing such pathogens. More specifically, the present invention
relates to methods and apparatus for concentrating pathogens in a
centrifugal chamber and for re-suspending them to permit withdrawal
of the concentrated pathogens from the chamber for testing,
quantifying and the like.
[0002] It is known to use centrifuges for the purpose of
concentrating water and food-borne microorganisms, particularly
pathogens including Clostridium, Streptococcus, Shigella,
Salmonella, and other species, as set forth in U.S. Pat. Nos.
5,961,846; 5,858,251; and 5,846,439, all of which are hereby
incorporated by reference into this description. These patents
disclose a technique for flowing large quantities of water or
fluidized foods through a semi-rigid belt channel in a blood
centrifuge, such as the IBM Model 2997 or the Cobe Spectra
centrifuge, to concentrate any microorganisms contained in the
fluid.
[0003] These centrifuges employ a disposable annular or
circumferential separation chamber that is mounted on a reusable
hardware platform. The centrifuge rotates the separation channels
as fluid flows through the channel, concentrating microorganisms
within the channel. In order to test, identify or otherwise
evaluate any pathogens or other microorganisms concentrated in the
channel at the end of the process, the disposable channel must be
removed from the centrifuge device, and any pathogens or other
microorganisms contained therein must be flushed from the
channel.
[0004] Although the centrifuges may work satisfactorily for
concentrating fluid-borne microorganisms, the steps of
re-suspending and removing concentrated pathogens from the
centrifuge separation channel have presented some difficulty, and
the above-identified patents describe a relatively complex
technique for recovery of the channel contents after the
centrifugation process has ended. First, according to the '439
patent, the separation channel is primed with water containing a
surfactant to enhance removal of the material later collected. In
addition, after the centrifugation is completed, and the contents
of the separation channel are drained into a beaker, the channel is
then cut in half and filled with a solution of surfactant. The cut
ends are clamped with Vise-Grips pliers, and the channel is shaken
vigorously and placed in a laboratory vortex to dislodge pathogens
that may have adhered to the inner walls of the channel. This
rinsing procedure is conducted several times, and the concentrate
and all the rinses are combined.
[0005] The disposable centrifuge channels used in the IBM 2997 and
COBE Spectra centrifuges are made of semi-rigid, somewhat brittle,
plastic material, which is not conducive to repeated flexing or the
like. This may have contributed to the difficulty in removing
concentrated pathogens from the channel and necessitated the use of
surfactant, Vise-Grip pliers and a laboratory vortex to aid in
removing the concentrated microorganisms. Also, the presence of
other residue in the channel may have made removal of the
microorganisms more difficult. The present invention is intended to
overcome one or more of the shortcomings associated with the prior
art devices and methods. As used in the following description and
claims, "fluid" (and formatives thereof) means any liquid,
excluding blood, blood cells, plasma or other blood components,
that flows sufficiently for continuous centrifugal processing, and
"pathogens" and "pathogenic organisms" (and formatives thereof)
mean a disease-causing or abnormality-causing organism and do not
include, in any event, blood cells such as red cells, white cells
and platelets.
SUMMARY
[0006] The present invention is generally embodied in method and
apparatus for concentrating and recovering pathogens from fluid by
employing a flexible centrifugation chamber, through which the
fluid is continuously flowed. The flexible centrifugation chamber
is subjected to centrifugal force by rotating the chamber about an
axis of rotation while fluid is being fed therethrough, so as to
concentrate in the chamber pathogens that may be contained in the
fluid. In accordance with the present invention, the flexibility of
the chamber enhances re-suspension of pathogens that are
concentrated therewithin, and the pathogens may be re-suspended by
shaking the flexible chamber with fluid contained therein. By
vigorously shaking the container to and fro, the fluid therein is
caused to slosh from end to end by virtue of the flexibility of the
chamber. This induces high shear stresses and promotes
re-suspension of the pathogens. The flexible chamber may also be
stretched such that the gap of the chamber can be adjusted. By
doing this, one can induce and control proper shear stress. This
can be done manually or automatically.
[0007] The step of shaking the chamber may be carried out manually
or automatically and may include squeezing and/or twisting of the
chamber to cause the fluid to slosh back and forth.
[0008] The flexible centrifugation chamber may be elongated, and
fluid may be introduced into the chamber substantially at one end
and withdrawn substantially at the other end of chamber.
Alternatively or additionally, the chamber may be subdivided into a
series of interconnected flow channel segments so that fluid
repeatedly substantially traverses the length or width of the
container as it passes therethrough, thereby decreasing stagnation
or unperfused areas of the chamber and resulting in a more uniform
flow field in the centrifugal field, and thus enhancing
concentration of pathogens in the chamber. Other serpentine flow
path arrangements may also be used within the flexible chamber.
[0009] In accordance with the present invention, a single
centrifuge chamber may be used in the concentration procedure.
Also, multiple chambers, formed of entirely separate chambers or
formed from a single disposable unit or chamber sub-divided into
two or more subchambers, may be employed for higher fluid
processing rates or collection efficiency. For example, the use of
separate containers or sub-chambers with separate inlets connected
in parallel to the fluid source may allow for higher processing
rates, since fluid is simultaneously being processing through two
chambers.
[0010] Also, separate chambers or sub-chambers may be connected in
series for improved efficiencies. The second chamber could be used
for the more specific collection of pathogens from the fluid. In
other words, the supernatant from the first stage will include many
of the target pathogens which can be concentrated in the second
chamber or stage. For particularly small pathogens, a sedimentation
enhancing agent, such as an affinity agent, for example, a chemical
enzyme, may be added to the supernatant from the first chamber to
enhance sedimentation of the pathogenic organisms contained
therein.
[0011] In a multiple stage or multiple chamber separation, the
first chamber in the series could be a simple plastic pouch, with
or without a simple u-shaped or other flow path, for collection of
a large volume of sediment. The second container could employ the
same or a lengthier flow path, such as shown in FIGS. 4a or 4b. In
either the parallel or series arrangement, one chamber (container)
could immediately be used for testing, identifying or quantifying
the pathogens, and the other chamber could be severed, sealed and
stored as an archive for future testing or reference if desired.
Additional chambers (more than two) also could be employed in
parallel or series in accordance with this aspect of the present
invention. Also, in the series arrangement, the first chamber could
include a passageway for withdrawal of concentrated particles
(which may include some of the pathogens) on an intermittent or
continuous basis.
[0012] The flexible centrifugation chamber may be fashioned in
various different ways without departing from the present
invention. In one preferred embodiment, the chamber is defined by a
pair of facing sheets of flexible plastic film that are sealed
together, as by heat or solvent, along at least a peripheral area
to define an interior chamber for centrifugal fluid processing.
Other forming techniques may also be used, provided that the end
result is a flexible centrifugation chamber that may be easily
deformed for re-suspension. For example, a rigid or semi-rigid
chamber could be used with a flexible liner. The rigid or
semi-rigid container could provide the desired shape for
centrifugation purposes, and the flexible liner removed after
centrifugation for easy re-suspension. Also, the chamber could be
partially rigid or semi-rigid and partially flexible. The areas of
the chambers where the pathogens concentrate could be made
flexible, and the remainder of the chamber or container could be
rigid or semi-rigid, which may be easier to shake.
[0013] In addition to the peripheral seal, other seal lines may be
provided between the facing plastic sheets to define an elongated
or serpentine flow path or to define a plurality of interconnected
flow channel segments within the chamber to potentially improve the
uniformity of the flow fields of fluid passing through the chamber
and enhance the collection efficiency. These additional seal lines
may be provided permanently by bonding together the facing plastic
sheets, as by heat or solvent bonding, or may be provided
temporarily by compressing the plastic sheets together in the
desired locations to form the desired flow path configuration
during centrifugation and allowing the films to separate to form a
single chamber after centrifugation is complete. These are but a
few of the features of the present invention found in the following
more detailed description.
DESCRIPTION OF DRAWINGS
[0014] FIG. 1 is a perspective view of a centrifuge that may be
used in the method of the present invention.
[0015] FIG. 2 is a flow diagram, illustrating the flow of fluid
through the centrifuge of FIG. 1 during the concentration
procedure, with the containers connected in a series
arrangement.
[0016] FIG. 3 is a perspective view of a clamp and platen
arrangement of the type used in the centrifuge shown in FIG. 1, for
cooperation with a disposable flexible container to temporarily
define a centrifugation chamber of selected configuration.
[0017] FIG. 4a is a plan view of the container depicted in FIG. 3,
illustrating one configuration of the chamber as defined by the
clamp and platen, in which the fluid flow path through the chamber
is generally U-shaped.
[0018] FIG. 4b is a plan view of the container depicted in FIG. 3,
illustrating another configuration of the chamber as may be defined
by the clamp and platen, in which the fluid flow path through the
chamber is generally serpentine, created by a series of
interconnected flow path segments that extend vertically in the
container.
[0019] FIG. 5 is a perspective view of another centrifuge that may
be used in accordance with the present invention.
[0020] FIG. 6 is a perspective view illustrating assembly of the
flexible centrifuge container on a center spool or core that is
mounted within the centrifuge of FIG. 5.
[0021] FIG. 7 is a cross-sectional view of the centrifuge
illustrated in FIG. 5, and showing the flexible container and
center spool mounted within an outer bowl for rotation within the
centrifuge.
[0022] FIG. 8 is a plan view of a flexible centrifuge chamber that
may be used in connection with the centrifuge shown in FIG. 5.
[0023] FIG. 9 is a perspective view of an alternative flexible
centrifuge chamber that may be used with the centrifuge of FIG.
5.
[0024] FIG. 10 is a perspective view of another alternative of the
flexible centrifuge chamber that may be used in combination with
the centrifuge of FIG. 5.
[0025] FIG. 11 is a diagrammatic view, depicting rotation of a
flexible centrifuge chamber in a centrifuge of a type generally
shown in FIG. 5.
[0026] FIG. 12 is a perspective view illustrating the to and fro,
or lengthwise shaking that may be used in the process of the
present invention to re-suspend pathogens that are concentrated in
the flexible centrifuge chamber.
DETAILED DESCRIPTION
[0027] FIG. 1 illustrates one type of centrifuge system that may be
used in carrying out the method of the present invention.
Specifically, FIG. 1 shows a CS-3000.RTM. centrifuge 20 of the type
that has long been manufactured and sold by the Fenwal Division of
Baxter Healthcare Corporation of Deerfield, Ill. The CS-3000
centrifuge system of FIG. 1 includes a reusable hardware portion 22
and a disposable tubing set or fluid circuit 24.
[0028] The centrifuge hardware portion includes a base 26, in which
the rotating portion of the centrifuge is located, and a control
panel 28, which contains pumps 30, valves and detectors (not shown)
and a user display and input section 32 for user control of the
centrifuge operation. As described in more detail in U.S. Pat. No.
4,525,515, which is hereby incorporated by reference into this
description, the disposable fluid circuit includes a control
housing or monitor box 34, through which the fluid tubing is
routed. The monitor box organizes the tubing for simplified
installation and mounts over sensors, valves and associated devices
on the control panel. Fluid flow tubing extends from and returns
into the housing 34 to form external tubing loops 35 adapted to fit
over a pair of peristaltic pumps 30, as seen in FIG. 1, for moving
fluid through tubing set 24.
[0029] Turning next to FIG. 3, the centrifuge of FIG. 1 employs a
pair of opposed clamps 36, which are orbited or rotated about an
axis of rotation. Each clamp holds a flexible plastic bag 38, which
forms a centrifuge chamber, which is part of the disposable fluid
circuit. The disposable tubing set has two such centrifuge bags,
one for each clamp.
[0030] The bags are typically in a series arrangement in the fluid
circuit, and fluid flows through the bags as illustrated in FIG. 2.
As shown there, fluid which may possibly be contaminated with
pathogens, flows from the fluid source through tubing into a first
one of the flexible centrifuge chambers formed from the bag 38. The
fluid exits that chamber and is directed into a second flexible
centrifuge chamber, from which it then exits for return to the
source or to a waste facility such as a drain or the like.
[0031] The flow rate of fluid through the centrifuge containers is
controlled by peristaltic pumps 30. Although two pumps are shown,
it is more likely that only one centrifuge pump would be used to
pump fluid from the source through both centrifuge chambers, when
in a series arrangement. Alternatively, the bags could be arranged
in parallel and each pump would draw fluid from a fluid source and
direct it through one of the bags for pathogen concentration. Such
a parallel processing arrangement could substantially reduce
processing time for a given quantity of source fluid.
[0032] In the CS-3000 centrifuge, each bag 38 is defined into the
desired centrifuge chamber shape by the respective clamp in which
it is mounted. Each clamp receives a platen 40 which has raised
surfaces designed to press against one side of the bag 38 to form
the bag into a selected shape. The same or different platens may be
used in each clamp, depending on the desired chamber
configuration.
[0033] As illustrated in FIG. 3, for example, the bag 38 which
forms the flexible centrifuge chamber is generally of the shape of
a flat pouch, formed by peripherally sealing together two facing
plastic sheets. The bag is located between hinged plates 42 of the
clamp 36. A platen 40 having raised surfaces of the desired
configuration, is also located between the hinged plates of the
clamp 36. When the clamp is closed, the platen presses the bag 38
against one side of the clamp, compressing the facing sheets of the
bag together in selected locations to form the desired
configuration for the centrifuge chamber.
[0034] An example of such a chamber configuration is shown in FIG.
4a. There, the facing sheets of the bag 38 are pressed together
along a vertical line 44 that extends from the upper peripheral
seal to a location spaced from the lower peripheral seal. This
forms a generally U-shaped flow path in the centrifuge chamber that
is defined by two vertical flow path segments 45 that extend the
length of the bag and are interconnected at the bottom gap between
the seal line 44 and the peripheral edge of the bag.
[0035] In accordance with a further alternative of the present
invention, the platen and clamp may be shaped to provide a series
of such vertical seal lines 44, as shown in FIG. 4b, extending
alternately from the upper and lower peripheral seals to define an
elongated, serpentine flow path of greater length defined by six
vertical flow path segments 45, thereby increasing the length of
the flow path and potentially enhancing removal of pathogens from
the fluid circulating therethrough by inducing a more uniform
perfusion.
[0036] Also, the shape and direction of the flow path could be
changed without departing from the present invention. The flow path
segments could extend horizontally, for example, or the flow path
could take other forms such as a spiral or circular arrangement to
increase the length of the flow path as desired.
[0037] After the concentration procedure is complete, the bag 38
may be removed from the clamp. In the absence of the clamping
pressure, the bag resumes its normal pouch-like configuration, free
of the vertical or other lines of compression, allowing the bag to
be vigorously shaken for improved re-suspension of pathogens
concentrated in the container.
[0038] FIG. 5 illustrates another type of centrifuge that may be
used in connection with the present invention. The centrifuge 46
shown there is the Amicus.RTM. centrifuge, which is made and sold
by Baxter Healthcare Corporation of Deerfield, Ill. The Amicus
centrifuge 46 also employs a reusable hardware portion 48, shown in
FIG. 5, and a disposable tubing set or fluid circuit 48, the
pertinent portion of which is shown in FIG. 6.
[0039] The reusable hardware portion 48 has a base 52, in which the
rotating parts of the centrifuge are contained, and an elevated
control screen and user input panel 54 for operator control of the
centrifuge operation. The base includes one or more pumping
stations 56 that are adapted to receive a flow control cassette and
various sensors and valves that cooperate with the tubing set for
controlling the flow of fluid through the disposable circuit.
[0040] The Amicus centrifuge was originally designed for separation
of blood and blood components, and employed three pumping stations
for controlling the flow of the different fluids, such as saline,
anticoagulant, whole blood, and blood complements through the fluid
circuit. It is contemplated that only one or two pumping stations
would be required for use of the Amicus centrifuge in connection
with the present invention, although the availability of additional
pumping stations adds flexibility for future applications that may
not be contemplated at the present time. The Amicus centrifuge and
associated disposable fluid circuit are described in more detail in
U.S. Pat. No. 5,547,453, which is hereby incorporated by reference
into this description.
[0041] As described more fully in the above-patent, the Amicus
centrifuge employs a spool and bowl arrangement in which an inner
spool 58 is located within an outer centrifuge bowl 60, and the
flexible centrifuge chamber, in the form of an elongated pouch or
belt 62, is located between the spool and bowl. FIG. 6 illustrates
mounting of a flexible centrifuge chamber, which is in the form of
a flexible plastic belt, around the outside of the spool 58. After
the flexible belt is mounted on the spool, the spool and belt are
placed within an outer centrifuge bowl 60. The spool and bowl are
in an inverted position, as shown in FIG. 6, when operating within
the centrifuge.
[0042] Fluid is introduced into the flexible centrifuge chamber and
withdrawn therefrom through a flexible umbilicus 64 that connects
the disposable centrifuge chamber to a stationary portion of the
centrifuge. As described more fully in the above-identified patents
and in U.S. Pat. No. 4,734,089, also incorporated by reference
herein, both the CS-3000 and Amicus centrifuges employ the 1w-2w
principle to provide a seal-less connection between the rotating
centrifuge chamber and the exterior of the centrifuge device. The
seal-less connection avoids the need for the rotating seal,
rotating seal lubrication, and the other assorted safeguards and
operational limitations associated with rotating seals in
high-speed centrifuges.
[0043] Various examples of the bag which may be used to define the
flexible centrifuge chamber in the Amicus centrifuge are shown in
FIGS. 8-10. As shown in FIG. 8, the chamber is defined by a plastic
web or belt formed by two flexible plastic sheets or films
peripherally sealed together, such as by heat or solvent bonding
commonly used to manufacture such containers in the medical
industry. A vertical seal line 66 divides the resulting pouch into
two sub-chambers or sub-pouches, which may be the same or different
size. An interior seal line 70 in each sub-pouch forms first and
second flow segments 72 and 74 in each sub-pouch, through which
fluid must flow. In the Amicus centrifuge, the seal lines are
typically permanent, and formed by heat or ultrasonic bonding of
the two facing plastic sheets.
[0044] The container (bag or belt) preferably has a sufficiently
thin wall and is made from a material sufficiently pliable to allow
ready flexing of the container walls by fluid sloshing within the
container during re-suspension. One example of such a material is
polyvinyl chloride (PVC) that has been plasticized with a selected
amount of a plasticizer such as DEHP or a citrate ester. Also the
interior surface of the facing sheets forming the belt may be
embossed to provide a slightly roughened surface. This serves to
prevent the sheets from adhering together and allowing separation
of the sheets when fluid is introduce. In addition the roughened
surface creates numerous microscopic barriers that may serve to
trap the very small pathogens and retard their movement along the
surface of the container and eventual re-entrainment in the fluid
circulating through the chamber. The result may be increased
pathogen capture and concentration efficiency.
[0045] As shown in FIG. 8, the seal line 70 is L-shaped, and has a
vertical portion that extends generally parallel to seal line 66
and a substantially horizontal portion that is spaced from the
lower edge of the belt and terminates just short of the end wall of
the belt to interconnect the flow segments and allow fluid to flow.
Fluid thus flows into an inlet 64 in each sub-pouch, through the
first segment 72, around the end of the horizontal seal line and
into and through the second segment 74 and through outlet 76. In
this arrangement, the fluid must flow substantially along the
length of the sub-chamber twice, and fluid cannot "short-cut"
between the inlet and outlet, which would reduce the residence time
in the centrifugal field and the concentration efficiency.
[0046] As discussed earlier in connection with the separate bags
used in the CS-3000 centrifuge, the subchambers of the Amicus
disposable belt may be connected in parallel or series and may be
of the same or different sizes. The subchambers may be free of any
interior seal, or additional seal lines may be used to create a
more uniform flow path or field within one or both flow chambers,
or any combination of these.
[0047] FIG. 9 is an alternative centrifuge chamber defined by a
flexible plastic belt 78 that is elongated, generally rectangularly
shaped, with an inlet 80 at one end and an outlet 82 at the other
end.
[0048] FIG. 10 illustrates yet a further embodiment a flexible
centrifuge chamber defined by a plastic bag or belt 84 in which
both inlet 86 and outlet 88 are at the same end of the flexible
plastic container, and an intermediate horizontal seal line 90
extends from one end of the container to a location spaced from the
other end to form the container into first and second flow
interconnected flow segments so that the fluid must traverse the
length of the container twice before exiting through the outlet. As
with the embodiment described in the CS 3000, additional
intermediate seal lines may be provided to define any desired
number of additional interconnected flow path segments so that
fluid passing through the bag is required to traverse the length or
width of the bag at least 4 and perhaps as many as 8 or more
times.
[0049] FIG. 11 is a diagrammatic illustration of the container of
FIG. 10 in the Amicus centrifuge. As shown there, the container 84
is located between an inner wall, which is defined by the spool 58,
and an outer wall which is defined by bowl 60, as best shown in
FIG. 7. Together they are rotated about an axis of rotation,
subjecting the bag and its contents to a centrifugal field which
tends to force the particles in the fluid, including pathogenic
organisms, toward the outermost wall of the container where they
can be concentrated.
[0050] In accordance with the method of the present procedure,
potentially contaminated fluid is flowed continuously through the
flexible centrifuge chamber or chambers located in the centrifuge.
As apparent from the illustrated examples, the centrifuge chamber
may be a single chamber, may be separate chambers, or may be a
single chamber that is subdivided into sub-chambers or sub-pouches.
Fluid may flow directly from inlet to outlet of the chamber or
through a lengthier, such as a serpentine channel, which requires
the fluid to traverse the length or width of the chamber 2 or more
times.
[0051] The centrifugal field or force selected may be the choice of
the user. Typically, however, it is believed that centrifugal field
generated by rotation of 1000-6000 rpm, with the centrifuge chamber
located at a radius of from about 1-6 inches from the axis of
rotation should provide sufficient centrifugal force to result in
concentration of pathogenic organisms that may be contained within
the fluid.
[0052] After a selected amount of fluid is processed through the
centrifugal chamber, the chamber, i.e., bag, belt or pouch, is
removed from the centrifuge, preferably but not necessarily with a
quantity of fluid contained therein. The chamber is then shaken
vigorously to and fro to re-suspend within the fluid any pathogenic
organisms that have been concentrated in the chamber. The highly
flexible and deformable container that is employed in both the CS
3000 and Amicus centrifuges allows the fluid therein, in effect, to
slosh back and forth from end to end, thus creating high shear
stresses that help re-suspend the pathogens that have been
concentrated within the chamber. Unlike the prior art semi-rigid
chambers of the IBM 2997 and Cobe Spectra centrifuges, it is
unnecessary to apply Vise-Grip pliers to the centrifuge chamber or
to subject the chamber to unique and time-consuming procedures to
re-suspend the pathogens that have been concentrated into the
container.
[0053] As shown in FIG. 12, the shaking of the container may be
carried out manually and may include twisting the container. During
re-suspension, the chamber (belt or bag) is held at both ends and
vigorously shaken to and fro (longitudinally). For example, shaking
the belt or bag to and fro can be achieved by grasping the belt or
bag at its ends (as generally depicted in FIG. 12) and shaking the
chamber from side to side, or holding the chamber in a vertical
position and shaking it up and down. Whether shaken horizontally,
vertically or in another direction ("to and fro" includes any of
these), the inertia of the fluid contained within the chamber tends
to concentrate the fluid in a central mass or bolus, as depicted by
the bulging flexible container walls in FIG. 12, that remains
essentially stationary as the walls of the bag move past, thereby
causing high shear stresses on the pathogens to help dislodge them
from the surfaces. Relative to the chamber, the fluid appears to
slosh back and forth one end to the other. In addition to the high
shear stresses established, the apparent sloshing causes the
flexible chamber to deform flexibly with each cycle of shaking and
may further help to dislodge the pathogens. Prior art rigid or
semirigid containers do not deform sufficiently for the fluid to
build into a central mass and thus cannot establish the high fluid
stress induced in the flexible chamber employed here.
[0054] The shaking could also be carried out automatically, and
different chamber configurations could be used to permit shaking.
For example, the centrifuge itself could be used to shake the bag
or belt that forms the chamber, or an external device could be
used. If the centrifuge itself is used to "shake" the bag for
re-suspension purposes (after the pathogen concentrated procedure
is completed), the centrifuge could employ a pneumatic device to
repeatedly push on the belt wall radially, sloshing the fluid back
and forth within the belt. For purposes of illustration, this could
be a pneumatic device or balloon or a series of such devices or
balloons located around the inside surface of the bowl in the
Amicus centrifuge, which could be rapidly and repeatedly inflated
against the belt wall to cause sloshing of fluid therein to
re-suspend the pathogens automatically. This also could be combined
with vibrating motion of the centrifuge, not necessarily along the
rotational axis, to aid in the re-suspension.
[0055] After shaking, the contents of the container are drained
into a beaker or other receptacle. Thereafter, rinse solution, such
as distilled water, may be added to the container, and the shaking
step repeated to insure that the re-suspended pathogenic organisms
are fully flushed from the container. Alternatively, distilled
water may be added to the container before the initial shaking to
re-suspend any pathogens concentrated in the container.
[0056] As pointed out earlier, separate processing chambers or
sub-chambers may be connected in series or parallel for better flow
rates and/or efficiencies, as well as to provide additional
features. In this regard, the first container in the series could
be simple pouch or employ a simple u-shaped flow path or the like,
and the second container could employ the same or a longer flow
path, such as shown in FIG. 4b for example. The first container in
this arrangement, which also may contain some concentrated
pathogens, could be severed from the fluid circuit, sealed and
stored as an archive for future reference if desired. A separate
withdrawal passageway may be provided in the first container for
withdrawing particles (which may include some pathogens)
concentrated therein. In such an embodiment, the umbilicus 64 could
include an additional passageway and one of the pumping stations 56
could be devoted to withdrawing particles from the container.
[0057] If chambers or subchambers are connected in the fluid
circuit in a parallel arrangement, processing time for a given
quantity of fluid may be significantly reduced over the series or
single chamber arrangements. This parallel arrangement also
provides the advantage of one chamber for immediate testing and a
second chamber which could be severed, sealed and stored for future
testing, verification or other purposes.
[0058] A series arrangement may have further advantages in
separating small pathogens. As illustrated in FIG. 2, after the
first chamber, a sedimentation or separation enhancing agent, such
as an affinity agent, for example, a chemical enzyme, may be added
to the supernatant from the first chamber (which potentially
contains the pathogens that the user desires to concentrate) to
enhance sedimentation of the pathogenic organisms during processing
in the second chamber or stage. Such an affinity agent could be
provided in a pre-attached container, as part of the disposable
fluid circuit with fluid flow tubing communicating between the
container and the fluid flow path between the first and second
chambers or stages, or the disposable fluid circuit could have a
facility such as an injection site or the like that permits user
addition of a selected affinity agent into the flow path.
Centrifuges with multiple pumps or pumping stations have the
flexibility to permit one of the pumps to be used for automatically
controlling the flow rate of such an affinity agent into the second
chamber or into the supernatant flow path upstream of the second
chamber according to a pre-selected or user-selected flow rate.
[0059] Although the present invention has been described in its
preferred and alternative embodiments, it is contemplated that
further alternatives will be apparent to one skilled in the field
upon reading this specification, and the that the scope of the
present invention is as defined in the appended claims, and not
limited to the features or details of the illustrated embodiments
unless expressly required by the appended claims.
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