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PARTIChE SEPARATION APPARATUS AND METHOD
Backctround of the Invention
Field of the Invention
The present invention relates to a method and_apparatus
for separating first pa--rticles from second particles and for
separating particles and liquid. The invention has~particular
advantages in connection with separating blood components.
Description of the Related Art
. In many different fields, liquids carrying particle
substances must be filtered or processed to obtain either a
purified liquid or purified particle end product. In its
broadest sense, a filter is any device capable of removing or
separating particles from a substance. Thus, the term
"filter" as used herein is not l,~mited to a porous media
material but includes many different types of processes where
particles are either separated from one another or from
liquid.
In the medical field, it is often necessary to filter
blood. Whole blood consists of various liquid constituents
and particle constituents. Sometimes, the particle
constituents are referred to as "formed elements". The liquid
portion of blood is largely made up of plasma, and the
particle constituents include red blood cells (erythrocytes),
white blood cells (including leukocytes), and platelets
(thrombocytes). While these constituents have similar
densities, their average density relationship, in order of
decreasing density, is as follows: red blood cells, white
blood cells, platelets, and plasma. In addition, the particle
.constituents are related according t.o size, in order of
decreasing size, as follows: white blood cells, red blood
cells, and platelets. The size of real blood cells, however,
may vary because red blood cells osmotically change size
depending on the hypotonicity or hypertonicity of a liquid,
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such as plasma, in which the red blood cells are disbursed-
When hypotonicity of plasma increases, the red blood cells
osmotically become larger. Conversely, when hypertonicity of
plasma increases, the red blood cells osmotically become
smaller. Most current purification devices rely on density w
and size differences or surface chemistry characteristics to
separate and/or filter the blood components.
Numerous therapeutic treatments require groups of
particles to be removed from whole blood before either liquid
or particle components can be infused into a patient. For
example, cancer patients often require platelet transfusions
after undergoing ablative, chemical, or radiation therapy. In
this procedure, donated whole blood is processed to remove
platelets and these platelets are then infused into the
patient. However, if a patient receives an excessive number
of foreign white blood cells as contamination in a platelet
transfusion, the patient's body may reject the platelet=
transfusion, leading to a host of serious health risks.
Typically, donated platelets are separated or harvested
from other blood components using a centrifuge. The
centrifuge rotates a blood reservoir to separate components
within the reservoir using centrifugal force. In use, blood
enters the reservoir while it is rotating at a very rapid
speed and centrifugal force stratifies the blood components,
so that particular components may be separately removed.
Centrifuges are effective at separating platelets from whole
blood, however they typically are unable to separate a7_1 of
the white blood cells from the platelets. Historically, blood
separation and centrifugation devices are typically unable to
consistently (990 of the time) produce platelet product that
meets the "leukopoor" standard of less than S x 106 white
blood cells for at least 3 x 1011 platelets collected.
Because typical centrifuge platelet collection processes
are unable to consistently and satisfactorily separate white
blood cells from platelets, other processes have been added to
improve results. In one procedure, after centrifuging,
platelets are passed through a porous woven or non-woven media
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filter, which may have a modified surface, to remove white
blood cells. However, use of the porous filter introduces its
own set of problems. Conventional porous filters may be
inefficient because they may permanently remove or trap
approximately 5-200 of the platelets. These conventional
filters may also reduce "platelet viability," meaning that
once passed through a filter a percentage of the platelets
cease to function properly and may be partially or fully
activated_ In addition, porous filters may cause the release
of brandykinin, which may lead to hypotensive episodes in a
patient. Porous filters are also expensive and often require
additional time consuming manual labor to perform a filtration
process.
Although porous filters are effective in removing a
substantial number of white blood cells, they have drawbacks.
For example, after centrifuging and before porous filtering, a
period of time must pass to give activated platelets time to
transform to a deactivated state. Otherwise, the activated
platelets are likely to clog the filter. Therefore, the use
of porous filters is not feasible in on-line processes.
Another separation process is one known as centrifugal
elutriation. This process separates cells suspended in a
liquid medium without the use of a membrane filter. In one
common form of elutriation, a cell batch is introduced into a
flow of liquid elutriation buffer. This liquid which carries
the cell batch in suspension, is then introduced into a
funnel-shaped chamber located in a spinning centrifuge. As
additional liquid buffer solution flows through the chamber,
the liquid sweeps smaller sized, slower-sedimenting cells
toward an elutriation boundary within the chamber, while
larger, faster-sedimenting cells migrate to an area of the
chamber having the greatest centrifugal force.
When the centrifugal force and force generated by the
fluid flow are balanced, the fluid flow is increased to force
slower-sedimenting cells from an exit port in the chamber,
while faster-sedimenting cells are retained in the chamber.
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If fluid flow through the chamber is increased, progressively
larger, faster-sedimenting cells may be removed from the
chamber.
Thus, centrifugal elutriation separates particles having
different sedimentation velocities. Stokes law describes
sedimentation velocity (SV) of a spherical particle as
follows:
2 r2 ~Pp - Pm) g
SV -
9
where, r is the radius of the particle, pp is the density of
the particle, pm is the density of the liquid medium, ~ is
the viscosity of the medium, and g is the gravitational or
centrifugal acceleration. Because the radius of a particle is
raised to the second power in the Stokes equation and the
density of the particle is not, the size of a cell, rather
than its density, greatly influences its sedimentation rate.
This explains why larger particles generally remain in a
chamber during centrifugal elutriation, while smaller
particles are released, if the particles have similar
densities.
As described in U.S. Patent No. 3,825,175 to Sartory,
centrifugal elutriation has a number of limitations. In most
of these processes, particles must be introduced within a flow
of fluid medium in separate discontinuous batches to allow for
sufficient particle separation. Thus, some elutriation
processes only permit separation in particle batches and
require an additional fluid medium to transport particles. In
addition, flow forces must be precisely balanced against
centrifugal force to allow for proper particle segregation.
Further, a Coriolis jetting effect takes place when
particles flow into an elutriation chamber from a high .
centrifugal field toward a lower centrifugal field. The fluid
and particles turbulently collide with an inner wall of the
chamber facing the rotational direction of the centrifuge.
This phenomenon mixes particles within the~chamber and reduces
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the effectiveness of the separation process. Further,
Coriolis jetting shunts flow along the inner wall from the
inlet directly to the outlet. Thus, particles pass around the
elutriative field to contaminate the end product.
Particle mixing by particle density inversion is an
additional problem encountered in some priorelutriation
processes. Fluid flowing within the elutriation chamber has a
decreasing velocity as it flows in the centripetal direction
from an entrance port toward an increased cross sectional
portion of the chamber. Because particles tend to concentrate
within a flowing liquid in areas of lower flow velocity,
rather than in areas of high flow velocity, the particles
concentrate near the increased cross-sectional area of the
chamber. Correspondingly, since flow velocity is greatest
adjacent the entrance port, the particle concentration is
reduced in this area. Density inversion of particles takes
place when the centrifugal force urges the particles from the
high particle concentration at the portion of increased cross-
section toward the entrance port. This particle turnover
reduces the effectiveness of particle separation by
elutriation.
In addition to red and white blood cells, plasma, and
platelets, human blood also includes other particle
components, such as T cells, stem cells, and, in some cases,
tumor cells. These cells have substantially similar
densities, but different sedimentation velocities and sizes.
Generally, stem cells are larger than T cells and smaller than
tumor cells. Some tumor cells (approximately 30%), however,
are smaller than stem cells.
Existing purification devices are capable of purifying
peripheral blood to isolate what is known as a peripheral cell
r
collection for transfusion or reinfusion purposes. The
peripheral cell collection typically includes primarily
plasma, red blood cells, stem cells, and T cells, and may also
include tumor cells if the donor's blood included such cells.
Although transfusion of a peripheral cell collection is a
common medical treatment, transfusion of a.large number of T
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cells or tumor cells into a patient may have adverse
consequences. Removal of T cells and tumor cells from a
peripheral cell collection or whole blood before transfusion,
however, is extremely difficult.
After undergoing a therapeutic treatment, such as
chemotherapy or radiation therapy to eliminate cancerous tumor
cells, cancer patients often receive a peripheral cell or bone
marrow transfusion to replace stem cells destroyed as a side
effect of the treatment. To reduce risks associated with
infusing blood components from a foreign donor, some of these
transfusions are autologous, where blood components collected
from the patient are later reinfused back to the patient.
Blood components initially collected from cancer patients
may include cancerous tumor cells, which are then infused back
into the cancer patient during reinfusion. This reinfusion of
tumor cells may diminish the effectiveness of therapeutic
treatments aimed at reducing cancerous tumors in a patient's
body.
Another type of transfusion, known as allogenic
transfusion, involves collecting blood components from a donor
and then infusing the collected blood components into a
recipient who is different from the donor. Sometimes,
however, the recipient of an allogenic transfusion experiences
a disease commonly known as graft versus host disease. In
graft versus host disease, T cells, which may accompany the
blood components, are infused into the recipient and
"recognize" that the recipient's body is "foreign" from that
of the donor's. These T cells "attack" healthy cells in the
recipient's body, rather than performing a normal
immunological protective function_
Prior attempts to separate stem cells from tumor cells or
separate stem cells from T cells prior to reinfusion have had
limited success. In one separation method, a selective
antibody is introduced into a collected blood component
sample. The selective antibody chemically attaches to stem
cells in the collection to "mark" them. To separate the
marked stem cells from the remaining blood.components, the
CA 02215986 2003-07-31
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CA 02215986 2003-07-31
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CA 02215986 2006-O1-10
-lla-
According to the present invention, there is also
provided an apparatus for separating particles from a
liquid, the apparatus comprising:
a fluid chamber (22, 122, 222, 322, 422, 522, 622, 722,
822a, 1022, 1022', 1122, 1122') having an inlet, an outlet, and a
wall extending between and connecting the inlet and the outlet,
the wall defining an interior, such that, the interior has a
maximum cross- sectional area at a position intermediate the
inlet and the outlet, and the wall converges from the position of
the maximum cross-sectional area toward the inlet;
a separation chamber (46, 546, 646, 746, 846) configured to
be received by a centrifuge rotor to centrifugally separate
particles within the separation chamber; and
means for fluid coupling the fluid chamber to the separation
chamber, the fluid coupling means being connected to the
separation chamber at a location permitting particles in the
separation chamber to pass into the fluid chamber, while
substantially preventing non-separated particles from passing
through the fluid coupling.
According to the present invention, there is also
provided a method of separating blood components, the
method comprising the steps of:
applying a centrifugal force to blood to separate the blood
into components including at least plasma, platelets, and white
blood cells,
whereby at least a portion of the platelets become at least
partially activated during the applying step; and
filtering the components in a fluid chamber (22, 122, 222,
322, 422, 522, 622, 722, 822a, 1022, 1022', 1122, 1122') to
remove white blood cells, while a substantial number of platelets
remain activated.
Preferably, according to the present invention,
there is also provided an apparatus for separating
particles from a liquid, the apparatus comprising:
CA 02215986 2006-O1-10
-11b-
means for initially separating within a separation chamber
(46, 546, 646, 746, 846) particles according to at least one of
different densities and different sedimentation velocities; and
means, including a fluid chamber (22, 122, 222, 322, 422,
522, 622, 722, 822a, 1022, 1022', 1122, 1122') fluidly coupled to
the initial separating means, for further separating particles
according to sedimentation velocity.
Preferably, according to the present invention,
there.is also provided 'a method of separating particles in
a liquid, the method comprising the steps of:
initially separating particles in a liquid according to at
least one of different densities and different sedimentation
velocities;
flowing at least a portion of the particles separated in the
initial separating step into a fluid chamber (22, 122, 222, 322,
422, 522, 622, 722, 822a, 1022, 1022', 1122, 1122'); and
further separating the particles within the fluid chamber
according to different sedimentation velocities.
According to the present invention, there is also
provided a method of separating blood components, the
method comprising the steps of:
rotating a centrifuge rotor (12, 512, 612, 812, 1134, 1134')
about an axis of rotation, the rotor having a separation chamber
(46, 546, 646, 746, 846) and fluid chamber (22, 122, 222, 322,
422, 522, 622, 722, 822a, 1022, 1022', 1122, 1122');
controlling rotation of the rotor;
conveying blood components into the separation chamber;
separating the blood components within the separation
chamber into at least first blood components and second blood
components;
directing second blood components and at least a portion of
the first blood components from the separation chamber into the
fluid chamber; and
filtering within the fluid chamber the first blood
components to remove at least a portion of first particles from
the second blood components.
CA 02215986 2003-07-31
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CA 02215986 2003-07-31
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CA 02215986 1997-10-03
WO 96/33023 PCT/U~96/05505
- 12 -
adjusted by adjusting the solute concentration thereby
adjusting the separation characteristics of the particles in
the fluid chamber. -
It is to be understood that both the foregoing general
description and the following detailed description are
exemplary, and are intended to provide further explanation of
the invention as claimed_ The accompanying drawings are
included to provide a further understanding of the invention
and are incorporated in and constitute a part of this
specification_ The drawings illustrate embodiments of the
invention and, together with the description, serve to explain
the principles of the invention.
Brief Description of the DrawincLs
Fig. 1 is a perspective view of a centrifuge apparatus in
accordance with a embodiment of the invention;
Fig. 2 is an exploded side view of a fluid chamber shown
in Fig. 1;
Fig. 3 is a partial schematic view of the apparatus of
Fig. 1 illustrating a detailed view of components of the
apparatus;
Fig. 4 depicts a portion of a tubing set in accordance
with the invention;
Fig. 5 schematically illustrates a saturated fluid.ized
bed formed within a fluid chamber, where the fluid chamber is
a component of the tubing set of Fig. 4 mounted to the
centrifuge apparatus of Fig. 1;
Fig. 6 is a cross-sectional view of a first alternate
embodiment of the fluid chamber of Fig. 2;
Fig. 7 is a cross-sectional view of a second alternative
embodiment of the fluid chamber of Fig. 2;
Fig. 8 is a cross-sectional view of a third alternative
embodiment of the fluid chamber of Fig. 2;
Fig. 9 is a cross-sectional view of a fourth alternative
embodiment of the fluid chamber of Fig. 2;
Fig. 10 is a perspective view of an alternate embodiment
of a centrifuge apparatus of the invention;
CA 02215986 2006-O1-10
-13-
Fig. 11 is a cross-sectional schematic view of another
embodiment of the centrifuge apparatus of the invention;
Fig. 12 is a partial perspective view of the centrifuge
apparatus of Fig . ~ 11;
Fig. 13 illustrates a separation chamber and fluid
chamber in another embodiment of the invention;
Fig. 14 is a partial view of a centrifuge apparatus
including a separation chamber and multiple fluid chambers in
a further embodiment of the invention;
Fig. 15 is a schec~iatic diagram illustrating a tubing set
in accordance with the present invention, including the
portion depicted in Fig. 4;
Fig. 16 is a cross-sectional view of a fifth alternative
embodiment of the fluid chamber of Fig. 2;
Fig. 17 is a cross-sectional view of a sixth alternative
embodiment of the fluid chamber of Fig. 16;
Fig. 18 is a schematic diagram of an embodiment of the
invention including a fluid supply,line coupled to a primary
substance container and additive containers;
Fig. 19 is a partial schematic diagram of a centrifuge
apparatus including a fluid chamber and supplemental fluid
chamber in accordance with an embodiment of the invention; and
Fig. 20 is a partial schematic diagram of a centrifuge
apparatus including a fluid chamber and separation channel in
accordance with the invention.
Description of the Preferred.Embodiments
Reference will now be made in detail to the present
preferred embodiments of the invention illustrated in the
accompanying drawings.
An embodiment of the present invention is described by
referring to its use with a COBE~ SPECTRA two stage sealless
blood component centrifuge manufactured by the assignee of the
invention. The COBE~ SPECTRA centrifuge incorporates a one-
omega/two-omega sealless tubing connection as disclosed in
U.S. Patent No. 4,425,112 to Ito. The COBE~ SPECTRATM
centrifuge also uses a two-stage blood component separation
CA 02215986 2006-O1-10
-14-
channel substantially as disclosed in U.S. Patent No.
4,708,712 to Mulzet. Although the embodiment of the
invention is described in combination with the COBE~
SPECTRAT''' centrifuge, this description is not intended to
limit the invention in any sense.
As will be apparent to one having skill in the art, the
present invention may be advantageously used in a variety of
centrifuge devices commonly used to separate blood into its
components. In particular, the present invention may be used
with any centrifugal apparatus that employs a component
collect line such as a platelet collect line or a platelet
rich plasma line, whether or not the apparatus employs a two '
stage channel or a one-omega/two-omega sealless tubing
connection.
In accordance with the invention there is provided an
apparatus for filtering first particles from a liquid,
comprising a centrifuge rotor coupled ~o a motor for rotating
the centrifuge rotor about an axis of rotation. As embodied
herein and illustrated in Fig. 1, centrifuge~l0 includes- a
w
rotor 12. The rotor 12 has an annular groove or passageway 14
having an open upper surface adapted to receive a conduit or
Channel 44 of a tubing set 70 shown in Fig. 4_ The passageway
14 completely surrounds the rotor's axis of rotation 13 and is
bounded.on an inner surface by wall 15 positioned on a top
surface 17 of rotor 12. A motor 16 is coupled to rotor 12 to
rotate the rotor 12 about the axis of rotation 13. This
coupling is accomplished directly or indirectly through a
shaft 18 connected to an arm 19 that mounts to the rotor 12.
Alternately, the shaft 18 may be coupled to the motor 16
through a gearing transmission (not shown). A shroud 20 is
positioned on the rotor 12 to protect the motor 16 and shaft
18.
In accordance with the present invention, a holder is
Provided for holding a fluid chamber on the rotor with an
outlet of__the fluid chamber positioned closer to the axis of
rotation than an inlet of the fluid chamber. As embodied
CA 02215986 1997-10-03
WO 96!33023 PCT/LTS96/05505
- 15 -
herein and as illustrated in Fig. 1, the holder may include a
mounting bracket 24 for maintaining a fluid chamber 22 on
4 rotor 12 with an outlet 32 generally positioned closer to the
rotation axis 13 than an inlet 28. The fluid chamber 22 fits
- within the mounting bracket 24 as illustrated in Fig. 1. The
fluid chamber 22 may also be secured to the rotor 12 at
alternate locations, such as beneath passageway 14. The fluid
chamber 22 may be constructed of a transparent or translucent
copolyester plastic, such as PETG, to allow viewing of the
contents within the chamber interior with the aid of an
optional strobe (not shown) during a centrifuge procedure.
As illustrated in Fig. 2, the fluid chamber 22 is formed
by joining a first chamber section 26 having the inlet 28 to a
second chamber section 30 having the outlet 32. The inlet 28
and outlet 32 are arranged along a longitudinal axis A-A.
In an embodiment of the invention the fluid chamber 22
has an interior volume of about 14.5 ml., although this
parameter-may-be--increased--or decreased depending on the
particular application. The interior of the first chamber
section 26 has a frustoconical shape with a conical angle 34a
of approximately 30 degrees. The interior of the second
chamber section 30 also has a frustoconical shape having a
conical angle 34b of approximately 120 degrees. These angles
may be varied. For example, the conical angle 34b may range
from approximately 90 to 120 degrees and the conical angle 34a
may range from approximately 30 to 90 degrees.
The volume of the fluid chamber 22 should be at least
large enough to accommodate enough platelets to provide a
saturated fluidized particle bed (described below) for a
particular range of flow rates, particle sizes, and centrifuge
rotor 12 speeds.
Preferably, the fluid chamber interior has a maximum
cross- sectional area 33 located at a position intermediate
the inlet 28 and outlet 32 where sections 26, 30 join. The
cross sectional area of the fluid chamber interior decreases
or tapers from the maximum cross-sectional area 33 in both
directions along axis A-A. Although the fluid chamber 22 is
CA 02215986 1997-10-03
WO 96/33023 PCT/LTS96/05505
- 16 -
depicted with two sections 26, 30 having frustoconical
interior shapes, the interior shapes may be paraboloidal, or
of any other shape having a major cross-sectional area greater
than the inlet or outlet area. The fluid chamber 22 may be
constructed from a unitary piece of plastic rather than from
separate sections.
In accordance with the present invention, there is also
provided means for supplying a substance to the inlet of the
fluid chamber. As embodied herein and as schematically
illustrated in Fig. 3, a pump 36 is fluidly connected to the
fluid chamber 22 through outflow tubing 38. The pump 36 draws
fluid and particles from the outlet 32 of the fluid chamber
22. The pump 36 is preferably a peristaltic pump or impeller
pump configured to prevent significant damage to blood
components, but any fluid pumping or drawing device may be
provided. In an alternative embodiment (not shown), the pump
36 may be fluidly connected to the inlet ofthe fluid chamber
22 to directly move substances into and through the fluid
chamber 22. The pump 22 may be mounted at any convenient
location.
In accordance with the invention there is also provided
means for controlling the motor and/or the supply means to
maintain a saturated fluidized bed of second particles within
the fluid chamber and to cause first particles to be retained
in the chamber. As embodied herein and illustrated in Fig. 3,
the controlling means may include a controller 40 connected to
both the centrifuge motor 16 and the pump 36. As explained in
detail below, during a centrifuge operation, controller 40
maintains a saturated fluidized particle bed within the fluid
chamber 22 to separate particles. Controller 40 may include a
computer having programmed instructions provided by a ROM or
RAM as is commonly known in the art.
The controller 40 may vary the rotational speed of the
centrifuge rotor 12 by regulating frequency, current, or
voltage of the electricity applied to the motor 16.
Alternatively, the rotor speed can be varied by shifting the
arrangement of a transmission (not shown),. such as by changing
CA 02215986 1997-10-03
WO 96/33023 PCT/US96/05505
- 17 -
gearing to alter a rotational coupling between the motor 16
and rotor 12_ The controller 40 may receive input from a
- rotational speed detector (not shown) to constantly monitor
the rotor speed.
The controller 40 may also regulate the pump 36 to vary
the flow rate of the substance supplied to the fluid chamber
22. For example, the controller 40 may vary the electricity
provided to the pump 36. Alternatively the controller 40 may
vary the flow rate to the chamber 22 by regulating a valving
structure (not shown) positioned either within an inflow
tubing 42 connected to the inlet 28 or within outflow tubing
38. The controller 40 may receive an input from a flow
detector (not shown) positioned within the inflow tubing 42 to
monitor the flow rate of substances entering the fluid chamber
22. Although a single controller 40 having multiple
operations is schematically depicted in the embodiment shown
in Fig. 3, the control means of the invention may include any
number of individual controllers, each for performing a single
function or a number of functions. The controller 40 may
control flow rates in many other ways as is known in the art.
As described above, the rotor 12 is configured with an
annular passageway 14 that is open along a top surface as
illustrated in Fig. 1. This passageway 14 is provided to
receive a channel 44 of tubing set 70, as partially shown in
Fig. 5. As best illustrated in Fig. 4, tubing set 70
preferably includes a semi-rigid conduit formed into a channel
44 having a generally rectangular cross-section. A connector
71 joins ends of the channel 44 to form an annular or loop
shape that fits within passageway 14. A supply line 78
provides whole blood to an inlet of the semi-rigid channel 44,
while a tubing segment 42, outlet lines 72, 74, and a control
line 76 allow for removal of blood components during a
centrifuge operation and flow control within the channel 44.
Further details of the general configuration and functioning
of the channel 44, tubing segment 42, and lines 72, 74, 76 and
78 are described in U.S. Patent 4,708,712.
CA 02215986 1997-10-03
WO 96/33023 PCT/ITa96/05505
- 18 -
A protective sheath 80 surrounds the lines 72, 74, 76, 78
and outflow tubing 38. When the channel 44 of the tubing set
70 is removably positioned within the passageway 14, the lines
72, 74, 76 and 78 extend through slots 82, 84, 86,
respectively, formed in wall 15, while the inflow tubing 42
rests in a slot 88 formed by passageway 14 (See Figs. 1 and
5) .
Fig. 15 is a more complete view of a second embodiment of
the tubing set 70. The tubing set 70 may further include a
plurality of additional elements for collecting blood
components including, but not limited to one or more donor
access lines 902, sample devices 904, spikes 906, filled
solution bags (not shown) for the addition of fluids to the
tubing set 70, wastes bags 908, accumulator bags 910, blood
component bags 912, pump cartridges 914 for interfitting with
various fluid pumps such as the pump 36, air chambers 916,
monitoring device interfaces 918, inter-connecting tubing and
fittings 920, and various miscellaneous elements and
accessories.
As shown in Figs. 3 and 5, a separation chamber 46 is
positioned within a flow passage of the channel 44. Particles
initially separate within the separation chamber 46 according
to density and/or sedimentation velocity in response to
centrifugal force. The separation chamber 46 includes a ridge
48 positioned on an outer wall of the passageway 14 for
deforming a portion of the channel 44 to create a dam 50
within the channel 44. Alternatively, the dam 50 may be a
permanent structure mounted within the flow passage of the
channel 44. Although only a single separation chamber 46 and
dam 50 are depicted in the figures, the flow passage may have
multiple separation chambers or dams depending upon desired
use.
When channel 44 is positioned in passageway 14, a collect
well 54 forms in channel 44 adjacent dam 50. A tubing segment
42 connecting an outlet 56 of the well 54 to the inlet 2.8 of
the fluid chamber 22 allows for separated substances in the
collect well 54 to be conveyed to the fluid.chamber 22.
CA 02215986 2006-O1-10
- 19 -
Although the embodiment shown in Figs. 3-5 includes a tubing ,
segment 42, any fluid coupling may be used between the
separation chamber 46 and fluid chamber 22. For example, the
inlet 28 of fluid chamber 22 may be directly connected to
channel 44.
A method of separating particles of blood is discussed
below with~reference to Figs. 3 and 5. Although the invention
is described in connection with a blood component separation
process, it should be.understood that the invention in its
broadest sense is not so limited. The invention may be used
to separate a number of different particles. In addition the
invention is applicable to both double needle and single
needle blood purification or filtration applications. For
example, the invention of the present application may be
practiced with the SINGLE NEEDLE RECIRCULATION SYSTEM FOR
HARVESTING BLOOD COMPONENTS of U.S. Patent No. 5,437,624,
issued August 1, 1995 .
Preferably the fluid chamber 22 is initially primed with
a low density fluid medium, such as air, saline solution, or
plasma, having a density less than or equal to the density of
liquid plasma. This priming fluid allows. for efficient
establishment of a saturated fluidized bed of platelets within
the fluid chamber 22. When a saline solution is used, this
liquid enters the channel 44 through supply line 78. The
saline then flows into the outlet 56 and through the chamber
22 when controller 40 activates pump 36. Controller 40 also
initiates operation of the motor 16 to rotate the centrifuge
rotor 12 and fluid chamber 22 in the direction of arrow "B" in
Fig.,3. During rotation, twisting of fluid lines 72, 74, 76,
78 and outflow tubing 38 connected to the centrifuge rotor 12
and fluid chamber 22 is prevented by a sealless one-omega/two-
omega tubing connection as is known in the art and described
in U.S. Patent No. 4,425,112.
After the apparatus is primed, and as the centrifuge
rotates, whole blood or blood components are introduced
through supply line 78 into the semi-rigid channel 44. When
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whole blood is used, the whole blood can be added to the~semi-
rigid channel 44 by transferring the blood directly from a
donor through supply line 78. In the alternative, the blood
may be transferred from a container, such as a blood bag, to
supply line 78.
The blood within the channel 44 is subjected to a
centrifugal force as the centrifuge rotor 12 continues to
rotate in the direction of arrow "B" in Fig. 3. This
centrifugal force acts in a radial direction away from the
axis of rotation 13 of the rotor 12 as indicated by.arrow "C"
in Fig. 3.
The blood components undergo an initial separation within
the channel 44. The components of whole blood~stratify in
order of decreasing density as follows: 1. red blood cells, 2.
white blood cells, 3. platelets, and 4. plasma. The
controller 40 regulates the rotational speed of the centrifuge
rotor 12 to ensure that this particle stratification takes
place. The blood particles form a huffy coat layer 58 and an
outer layer 60 along an outer wall surface of the channel 44
within the separation chamber 46. The outer layer 60 includes
particles having a density greater than the density of the
particles in the huffy coat layer 58. Typically, the outer
layer 60 includes red blood cells and white blood cells, while
the huffy coat layer 58 includes platelets and white blood
cells . __
Plasma, the least dense blood component, flows within the
channel 44 along the top surface of the huffy coat layer 58.
When the height of the huffy coat layer 58 approaches the top
of dam 50, the flowing plasma washes the platelets and some
white blood cells of the huffy layer 58 over the dam S0.
After these particles are washed over the dam 50, they enter
the collect well 54. Some of the platelets may also flow past
the collect well 54 and then reverse direction to settle back
into the collect well 54,
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The white blood cells and red blood cells within the
outer layer 60 are removed through outlet line 74, while " .,
platelet poor plasma is removed through outlet line 72. The
controller 40 may control optional pumps (not shown) connected
to lines 72, 74, or 76 to remove these blood components, as is
known in the art. After the red blood cells, white blood
cells, and plasma.are thus removed, they are collected and
recombined with other blood components or further separated.
Alternately, these removed blood components may be reinfused
into a donor.
Plasma carries platelets and white blood cells from the
collect well 54 into the fluid chamber 22, filled with the
priming fluid, so that a saturated fluidized particle bed may
be formed. The controller 40 maintains the rotation speed of
the rotor 12 within a predetermined rotational speed range to
facilitate formation of this saturated fluidized bed. In
addition, the controller 40 regulates the pump 36 to convey
plasma, platelets, and white blood cells at a predetermined
flow rate through the tubing segment 42 and into inlet 28 of
the fluid chamber 22. These flowing blood components displace
the priming fluid from the fluid chamber 22.
When the platelet and white blood cell particles enter _.
the fluid chamber 22, they are subjected to two opposing
forces. Plasma flowing through the fluid chamber with the aid
of pump 36 establishes a first viscous drag foxce when plasma
flowing through the fluid chamber 22 urges the particles
toward the outlet 32 in the direction "D", shown in Fig. 3. A
second centrifugal force created by rotation of the rotor 12
and fluid chamber 22 acts in the direction "C" to urge the
particles toward the inlet 28.
The controller 40 regulates the rotational speed of the
rotor 12 and the flow rate of the pump 36 to collect platelets
and white blood cells in the fluid chamber 22. As plasma
flows through the fluid chamber 22, the flow velocity of the
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plasma decreases as the plasma flow approaches the maximum
cross-sectional area 33. This flow reaches a minimum velocity
at this maximum cross-sectional area 33. Because the rotating
centrifuge rotor 12 creates a sufficient gravitational field
in the fluid chamber 22, the platelets accumulate near the
maximum cross-sectional area 33 rather than flowing from the
fluid chamber 22 with the plasma. The white blood cells
accumulate somewhat below the maximum cross- sectional area
33. However, density inversion tends to mix these particles
slightly during this initial establishment of the saturated
fluidized particle bed.
The larger white blood cells accumulate closer to inlet
28 than the smaller platelet cells, because of their different
sedimentation velocities. Preferably, the rotational speed
and flow rate are controlled so that very few platelets and
white blood cells flow from the fluid chamber 22 during
formation of the saturated fluidized particle bed.
The platelets and white blood cells continue to
accumulate in the fluid chamber 22 while plasma flows through
the fluid chamber 22. As the concentration of platelets
increases, the interstices between the particles become
reduced and the viscous drag force from the plasma flow
gradually increases. Eventually the platelet bed becomes a
saturated fluidized particle bed within the fluid chamber 22.
Since the bed is now saturated with platelets, for each new
platelet that enters the saturated bed in the fluid chamber
22, a single platelet must exit the bed. Thus, the bed
operates at a steady state condition with platelets exiting
the bed at a rate equal to the rate additional platelets enter
the bed after flowing through inlet 28. This bed is depicted
schematically in Fig. 5, where the "X" symbol represents
platelets and the "O" symbol represents white blood cells. As
explained below, and depicted in Fig. 5, the saturated
fluidized particle bed substantially obstructs or prevents
white blood cells, "O", from passing through the fluid chamber
22.
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The saturated bed establishes itself automatically,
independent of the concentration of particles flowing into the
fluid chamber 22. Plasma flowing into the fluid chamber 22
passes through the platelet bed both before and after the
platelet saturation point.
The saturated bed of platelets occupies a varying volume
in the fluid chamber 22 near the maximum cross-sectional area
33, depending on the flow rate and centrifugal field. The
number of platelets in the saturated bed depends on a number
of factors, such as the flow rate into the fluid chamber 22,
the volume of the fluid chamber 22 and rotational speed. If
these variables remain constant, the number of platelets in
the saturated f-luidized bed remains substantially constant.
When the flow rate of blood components into the fluid chamber
22 changes, the bed self adjusts to maintain itself by either
releasing excess platelets or accepting additional platelets
flowing into the fluid chamber 22. For example, when the
plasma flow rate into the fluid chamber 22 increases, this
additional plasma flow sweeps excess platelets out of the now
super-saturated bed, and the bed reestablishes itself in the
saturated condition at the increased flow rate. Therefore,
the concentration of platelets in the bed is lower due to the
release of bed platelets.
After the saturated fluidized bed of platelets forms,
flowing plasma carries additional platelets into the fluid
chamber 22 and the bed.- These additional platelets add to the
bed and increase the viscous drag of the plasma flow through
the bed. At some point the viscous drag is sufficient to
cause platelets near the maximum cross-section area 33 to exit
the saturated bed and fluid chamber 22. Thus, if the
rotational speed and flow rate into the fluid chamber 22
- remain constant, the number and concentration of platelets
flowing into the saturated fluidized bed of platelets
substantially equals the number and concentration of platelets
released from the bed. This is in sharp contrast from the
prior art.
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Although the bed is saturated with platelets, a small
number of white blood cells maybe interspersed in the
platelet bed. These white blood cells, however will tend to
"fall" or settle out of the platelet bed toward inlet 28 due
to their higher sedimentation velocity. Most white blood
cells generally collect within the fluid chamber 22 between
the saturated platelet bed and the inlet 28, as depicted in
Fig. 5 and described below.
The saturated fluidized bed of platelet particles
functions as a filter or barrier to white blood cells flowing
into the fluid chamber 22. When blood components flow into
the fluid chamber 22, plasma freely passes through the bed.
However, the saturated fluidized platelet bedcreates a
substantial barrier to white blood cells entering the fluid
chamber 22 and retains these white blood cells within the
fluid chamber 22. Thus, the bed effectively filters white
blood cells from the blood components continuously entering
the fluid chamber 22, while allowing plasma and platelets
released from the saturated bed to exit the chamber 22. This
replenishment and release of platelets is referred to a.s the
bed's self-selecting quality. Substantially all of these
filtered white blood cells accumulate within the fluid chamber
22 between the saturated fluidized platelet bed and the inlet
28.
The particle separation or filtration of the saturated
fluidized particle bed obviates a number of limitations
associated with prior art elutriation. For example, particles
may be separated or filtered in a continuous steady state
manner without batch processing. In addition, an additional
elutriating fluid medium is not required. Furthermore, after
the saturated fluidized particle bed is established, flow
rates may be varied over a range without changing the size of
the particles leaving the fluid chamber 22. Unlike prior art
elutriation, the present invention establishes a saturated
particle bed consisting of numerically predominant particles.
This bed automatically passes the predominant particles while
rejecting larger particles.
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The apparatus and method of the invention separate
substantially all of the white blood cells from the platelets
- and plasma flowing through the fluid chamber 22. The barrier
to white blood cells is created, at least in part, because
white blood cells have a size and sedimentation velocity
greater than that of the platelets forming the saturated
fluidized particle bed. Therefore, particles of similar
densities are separated according to different sizes or
sedimentation velocities.
Because the initial separation at dam 50 and the
saturated fluidized bed remove a majority of the red blood
cells and white blood cells, the fluid exiting the fluid
chamber 22 consists mainly of plasma and platelets. A device
as described herein was operated 30 times using whole human
blood. Each operation resulted in a leukopoor product having
less than 3x106 white blood cells per 3x1011 platelets. Based
on these results it is expected that platelet product exiting
the fluid chamber 22 will consistently (at least 990 of the
time) meet the leukopoor standard of less than 5 x 106 white
blood cells when at least 3 x 1011 platelets flow from the
fluid chamber 22.
Unlike a conventional porous filter, where the filtered
white blood cells are retained in the filter, the present
invention allows a substantial fraction of white blood cells
to be recovered and returned to the donor.
Preferably, 80o to 990 of the platelets initially
entering the channel 44 may be recovered in a viable state.
More preferably, at least 950 or at least 980 of the platelets
initially entering the channel 44 are recovered from both the
channel 44 and the fluid chamber 22.
When the blood components are initially separated with
the separation chamber 46, a substantial number of platelets
may become slightly activated. The saturated fluidized
platelet bed allows white blood cells to be filtered from
plasma and platelets despite this slight activation. Thus,
the present invention does not require a waiting period to
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filter white blood cells after blood components undergo
initial separation in a separation chamber 46_ This is in
contrast to methods using conventional filters.
After separation, the platelets and plasma exiting the
fluid chamber 22 are collected in appropriate containers and
stored for later use. The red blood cells and white b7_ood
cells removed from the semi-rigid channel 44 may be combined
with the remainder of the plasma in the system for donor
reinfusion or storage. Alternatively, these components may be
further separated by the apparatus 10.
In an embodiment of the invention, the controller 40
regulates the rotational speed of the rotor 12 within a
preferred range of 1,800 to 2,400 RPM. Preferably the
rotation is controlled at 2,400 RPM to create a gravitational
field within the fluid chamber 22 ranging from approximately
8006 adjacent to the inlet 28 to approximately 5006 adjacent
to the outlet 32. The controller 40 maintains the flow rate
into the fluid chamber 22 within a range of 1 ml/min to 15 ml/
min. The preferred flow rate ranges from 2 ml/min to 8 ml/
min. The specific flow rate is selected according to an
initial platelet count and the total volume of whole blood
being processed, among other things.
In an embodiment of the invention, filtering may take
place at the same flow rate used to form the saturated
fluidized bed. Optionally the flow rate during filtering may
be greater than the flow rate used during the bed formation to
increase the rate of particle filtration. For this optional
arrangement, the controller 40 may increase the flow rate of
the blood components entering the fluid chamber 22 while
maintaining the saturated fluidized bed_ The controller 40
increases flow rate by increasing the rate of pump 36.
The controller 40 maintains the saturated fluidized bed
throughout the filtering process as blood components flow into
the fluid chamber 22. The controller 40 ensures that the flow
through the fluid chamber 22 is smooth and steady by
regulating the supply of fluid and rotation of the centrifuge
rotor 12. This regulation properly balances the forces in the
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fluid chamber 22 to maintain the saturated fluidized bed. As
described in more detail below, the controller 40 may increase
- flow into the fluid chamber 22 while maintaining the saturated
fluidized bed.
At the end of a blood component separation session, the
controller 40 may recover platelets retained both in the buffy
coat layer 58 of channel 44 and within the saturated fluidized
bed of fluid chamber 22. The controller-40 recovers platelets
in the buffy coat layer 58 by either decreasing rotational
speed of the rotor 12 or increasing the amount of plasma
exiting the channel 44. For example, in a preferred manner of
recovering platelets in the buffy coat layer, the rotor speed
is suddenly decreased from 2,400 RPM to 1,800 RPM, and then
increased back to 2,400 RPM. This spills platelets and white
blood cells retained in the buffy coat layer 58 over the dam
50 and into the fluid chamber 22. Within the fluid chamber 22
the saturated fluidized platelet bed blocks passage of the
white blood cells from the buffy coat layer 58, while buffy
coat layer 58 platelets simultaneously add to the bed and
release platelets from the saturated bed. Thus, the apparatus
may filter substantially all of the white blood cells from the
buffy coat layer 58. This increases platelet yield
significantly.
The buffy coat layer 58 may spill over the dam in the
manner described in above-mentioned U.S. Patent Application.
08/422,598. This is particularly effective in a mononuclear
cell collection procedure, because the fluid chamber 22 may
allow for separation of red blood cells from mononuclear
cells.
In addition, platelets in the saturated fluidized bed are
harvested to recover a substantial number of platelets from
the fluid chamber 22. During bed harvest, the controller 40
increases the flow rate and/or decreases the centrifuge
rotator 12 speed to release platelets from the bed. This
flushes from the fluid chamber 22 most of the platelets that
made up the saturated fluidized bed to substantially increase
platelet yield. The harvesting continues until substantially
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all of the platelets are removed, just before an unacceptable
number of white blood cells begin to flow from the fluid
chamber 22.
The harvested platelets that made up the bed may be
combined with the platelets previously collected. In
addition, the remainder of contents of the fluid chamber 22,
having a high concentration of white blood cells, can be
separately collected for later use or recombined with the
blood components removed from channel 44 for -return to a
donor.
The invention particularly allows trace or contaminating
first particles to be separated from a liquid having a larger
number of second particles. Preferably the first particles to
be filtered, such as white blood cells, have a concentration
insufficient to form a saturated fluidized particle bed.
However, the invention in its broadest application is directed
to either separating first particles from liquid or separating
first from second particles without concern for particular
particle concentrations.
Although the inventive device and method have been
described in terms of removing white blood cells and
collecting platelets, this description is not to be construed
as a limitation on the scope of the invention. The invention
may be used to separate any of the particle components of
blood from one another. For example, the saturated fluidized
bed may be formed from red blood cells to prevent flow of
white blood cells through the fluid chamber 22, so long as the
red blood cells do not rouleau (clump). Alternatively, the
liquid for carrying the particles may be saline or another
substitute for plasma. In addition, the invention may be
practiced to remove white blood cells or other components from
whole blood removed from an umbilical cord to collect stem
cells. Further, one could practice the invention by filtering
or separating particles from fluids unrelated to either blood
or biologically related substances.
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The apparatus and method of the invention may separate
white blood cells, including stem cells, and tumor cells by
- forming a saturated fluidized particle bed of stem cells to
substantially prevent tumor cells from flowing through the
r fluid chamber 22. In the alternative, the tumor cells may
form a saturated fluidized particle bed to substantially
obstruct flow of stem cells through the fluid chamber 22.
In another aspect of the invention, smaller first
particles, such as tumor cells, may be separated from larger
second particles, such as stem cells, by forming a saturated
fluidized particle bed with intermediate sized third
particles. Initially, intermediate sized third particles are
added to a liquid carrying the first and second particles.
Preferably, the concentration of added third particles exceeds
the concentration of both the first and second particles.
These third-particles are preferably magnetic micro-beads or
some other substance readily separable from the other
particles.
The liquid carrying the first, second, and third
particles then passes into the fluid chamber 22. Eventually,
the third particles form a saturated fluidized particle bed,
in the same manner as described above. As more of the liquid
and particles flow into the fluid chamber 22, the liquid and
smaller first particles pass through the saturated third
particle bed, while the bed and particle sedimentation
characteristics obstruct movement of the second particles
through the bed. Thus, the first and second particles
separate within the fluid chamber 22.
The saturated fluidized bed may release third particles
as more third particles flow into the bed or when flow rate
into the fluid chamber 22 changes. These third particles may
be removed from the liquid and first particles exiting the
fluid chamber 22. In a preferred embodiment, a particle
remover (not shown) having a magnet, magnetically attracts
magnetic third particles to remove them from the liquid.
Thus, a substantially purified concentration of first
particles is obtained.
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This alternate method is useful to separate first and
second particles both being present in low concentrations and
having similar densities, but different sizes. Although the
third particles for forming the bed are preferably added along
with the first and second particles, they may also be
introduced into the fluid chamber 22 in separate steps. This
aspect of the invention may be particularly useful in
separating tumor cells from stem cells or other blood
components, as mentioned above_ Alternatively, the first
particles may be T cells and the second particles may be stem
cells. However, this variant of the inventive method may be
practiced to separate many different types of particles. For
example, T cells can be separated from stem cells so as to
reduce graft versus host disease after stem cell transfusion.
Additional embodiments of the invention will now be
described where like or similar elements are identified
throughout the drawings by reference characters having the
same final two digits.
As shown in Fig. 6, another embodiment of the invention
includes a fluid chamber 122 having an inlet 128 and an outlet
132. A groove 190 is formed on an inner surface of the fluid
chamber 122 at a position of the maximum cross-sectional area
133. Top and bottom portions 191, 192, oriented substantially
perpendicular to a longitudinal axis A-A of the fluid chamber
122, are joined by a side 193. Preferably, the side 193 is
parallel to the axis A-A and surrounds this axis~to form the
substantially annular groove 190.
In an embodiment of the invention, the side 193 is 0.1
inches, while the top and bottom portions 191, 192 are each
0.08 inches. However, the groove 190 may be configured in
many different shapes and sizes without departing from the
invention.
The groove 190 helps to disperse Coriolis jetting within
the fluid chamber 122. Thus, groove 190 improves the particle
barrier capability of the saturated fluidized particle bed.
Sudden increases in liquid flow rate during a particle
separation procedure may limit the ability.of the saturated
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fluidized particle bed to obstruct particle passage. Liquid
flowing into the fluid chamber 22 undergoes a Coriolis jetting
' effect. This jetting flow reduces the filtration
effectiveness of the saturated fluidized particle bed because
' liquid and particles may pass between the saturated fluidized
particle bed and an interior wall surface of the fluid chamber
22 rather than into the bed itself. The fluid chamber 122
including groove 190 counteracts these effects by channeling
Coriolis jetting flow in a circumferential direction partially
around the axis A-A of fluid chamber 122. Therefore, the
groove 190 improves the particle obstruction capability of the
saturated bed, especially when liquid flow rates increase.
Figs. 16 and 17 depict fluid chambers 1022 and 1022',
respectively, having alternate embodiments of grooves 1090,
1090'. As illustrated in Fig. 16, fluid chamber 1022 includes
a groove 1090 having a side 1093 and top and bottom portions
1091 and 1092 formed circumferentially on the inner surface of
the fluid chamber 1022 at a position of a maximum cross-
sectional area 1033. These top and bottom portions 1091 and
1092 may be perpendicular to a longitudinal axis A-A, while
the side 1093 may be parallel to the axis A-A to form a
substantially annular groove 1090.
As shown in Fig. 16, a circumferential lip 1094, located
closer to the axis A-A than the side 1093, extends from the
top portion 1091. The bottom portion 1092 and lip 1094 define
a groove entrance 1096. As shown in Fig. 16, this groove
entrance 1096 may completely surround axis A-A.
Alternatively, as shown in Fig. 17, the groove entrance
may include a plurality of slot-shaped entrances 1095' spaced
about the circumference of the fluid chamber 1022' at the
position of maximum cross-sectional area 1033'. In this
embodiment, the lip 1094' extends to the bottom portion 1092'
to form an inner groove wall 1097' located between the slot-
shaped entrances 1095.
Preferably, a first entrance 1095' may be provided at a
location corresponding to the location of Coriolis jetting,
and a second entrance (not shown) may be provided at a
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diametrically opposite location. The Coriolis jet flow enters
the groove 1090' at a first slot-shaped entrance 1095',
travels circumferentially around the groove 1090' in both '
clockwise and counter-clockwise directions, and then exits at
another slot-shaped opening.
The configurations of Figs. 16 and 17 are believed to
improve direction of Coriolis jetting momentum and further
improve performance. The groove configurations of Figs. 16
and 17 may be optionally employed in conjunction with any of
the fluid chamber embodiments described herein.
Fig. 7 depicts another embodiment of a fluid chamber 222.
A plurality of steps 294 are formed on an inner-surface of the
fluid chamber 222 between the position of the maximum cross
section 233 and the inlet 228. Although only four steps 294
are illustrated, any number of steps 294 may be provided in
the fluid chamber 222.
Each step 294 has a base surface 295 oriented
substantially perpendicular to a longitudinal fluid chamber
axis A-A_ In addition, a side surface 296 is positioned
orthogonal to the base surface 295. Although Fig. 7 depicts a
corner where side surface 295 and base surface 295 intersect,
a concave groove may replace this corner. In a preferred-
embodiment each step 294 surrounds the axis A-A to bound a
cylindrical shaped area. Further, the fluid chamber 222
optionally includes a groove 290.
The base surface 295 is 0.05 inches and the side surface
296 is 0.02 inches in a preferred embodiment. However, the
sizes for these surfaces and the configuration of each step
294 may be modified without departing from the scope or spirit
of the invention.
Adding steps 294 to the fluid chamber 222, also improves
the particle obstruction characteristics of the saturated
fluidized particles bed, in particular during increases in the
rate of fluid flow. The steps 294 provide this improvement by
providing momentum deflecting and redirecting surfaces to
reduce Coriolis jetting in fluid chamber 222. When Coriolis
jetting takes place, the liquid and particles of the jet
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travel along an interior surface of the fluid chamber 222 that
faces the direction of centrifuge rotation. Therefore, the
- jet may transport particles between the fluid chamber interior
surface and either a saturated fluidized particle bed or an
elutriation field positioned in the fluid chamber 222. Thus,
particles traveling in the jet may exit the fluid chamber 222
without being separated.
Steps 294 direct or alter the momentum of the Coriolis
jet flow of liquid and particles generally in a
circumferential direction about axis A-A. Thus, a substantial
number of particles originally flowing in the jet must enter
the saturated fluidized bed or elutriation field to be
separated.
As shown in Fig. 7, the fluid chamber 222 may include
additional steps 225 shaped similar to steps 294. The
additional steps 225 are located between the position of the
maximum cross- section 233 and a fluid chamber outlet 232. In
a fashion similar to that described above, these steps 225
tend to redirect the Coriolis jet flow in a circumferential
direction surrounding axis A-A.
In addition to adding steps 294 and 225, the conical
angle 234b of the second chamber section 230 may be decreased
from 120° to 45° to reduce particle contamination caused by
density inversion. If faster sedimenting particles should
migrate past the maximum cross section 233, the smaller angled
walls partially limit some of these particles from flowing
directly to outlet 232 because density inversion does not
exist in the section 230. Thus, the faster sedimenting
particles will ~~fall" or migrate back between area 233 and
inlet 228 under the influence of the gravity centrifugal
field, rather than flowing from outlet 232. Optionally, any
of the fluid chambers disclosed herein may include a second
chamber section 230 with a conical angle 234b less than 120°.
Fig. 8 illustrates an additional embodiment of a fluid
chamber 322 having steps 394 and an optional groove 390
similar to groove 190. As shown in Fig. 8, each step 394
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includes a base surface 395 substantially perpendicular to
axis A-A. This embodiment also includes a side surface 396
oriented at an acute angle to the base surface 395. -
In Fig. 9 a further embodiment of a fluid chamber 422
including flow limiting steps 494 and an optional groove 490
is depicted. A side surface 496 of each step 494 is
substantially parallel to an axis A-A and forms an obtuse
angle with base surface 495.
In Figs. 6-9; the grooves 190, 290, 390, 490 and steps
294, 394, 494 may either completely or only partially surround
the chamber axis A-A. These features are provided to
facilitate fluid flow rate increases, as described below, as
well as to improve steady state performance of the fluid
chamber. During blood component separation, the grooves 190,
290, 390, 490 and steps 294, 394, 494 greatly reduce th.e
number of white blood cells that would otherwise bypass the
saturated fluidized platelet bed.
The grooves 190, 290, 390, 490, steps 294, 394, 494, and
additional steps 225 may be formed in many different
configurations without departing from the scope, or spirit of
the invention. For example, the base surface and/or side
surface of one or more of the steps 294, 394, 494 may have a
concave shape. In addition the steps 294, 394, 494 may be
arranged in a spiral surrounding axis A-A. Further, a portion
of the base surface 295, 395, 495 extending around axis A-A
may be positioned lower than a remainder of this surface to
form a concavity.
Although the fluid chambers 122, 222, 322, 422, 1022,
1022' are described for use in forming a saturated fluidized
particle bed, this description is not intended to limit the
scope of the invention in its broadest sense. Because
particle separation difficulties, such as those discussed in
Sartorv, may be reduced or eliminated, a significant
advancement from the prior art has been made. These fluid
chambers 122, 222, 322, 422 may be used in any particle
separation process. In particular, a fluid chamber having a
channel, step, or additional step may be used in elutriation.
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The grooves 190, 290, 390, 490, steps 294, 394, 494, and
additional step 225 may be formed in an injection molding
process. Preferably, the fluid chambers 22, 122, 222, 322,
422, 1022, 1022' are initially formed by injection molding in
multiple pieces, preferably two, and bonded together by any of
several well known processes, such as RF welding, ultrasonic
welding, hot plate welding, solvent bonding, or adhesive
bonding. Alternatively, these fluid chambers may be formed
from a unitary plastic material, as by blow molding. However,
any known manufacturing process may be practiced to fabricate
the chambers.
When one of the fluid chambers 122, 222, 322, 422, 1022,
1022' is substituted for the fluid chamber 22, the controller
40 may regulate flow of the liquid having first particles in
number of preferred ways. Because these fluid chamber designs
reduce Coriolis jetting and/or density inversion, the
controller 40 may increase flow rate without disrupting the
saturated fluidized particle bed.
While maintaining rotational speed of the rotor 12 at a
substantially constant rate, the controller 40 may increase
flow through the fluid chamber 122, 222, 322, 422, 1022,
1022' with one of, or a combination of, the following
different routines. In one routine the controller 40
increases flow rate by rapidly or instantaneously increasing
flow through the fluid chamber 122, 222, 322, 422, 1022,
1022'. In another routine, flow rate is increased gradually
over time. In yet a further routine, the controller 40
increases flow rate in a sequential manner by gradually
increasing the flow rate, maintaining this increased flow
rate, and then gradually increasing flow rate again.
However, if the apparatus 10 includes the fluid chamber
22, shown in Figs 1-5, the flow rate control may be more
limited. The flow velocity of the liquid and particles
entering the fluid chamber 22 should not undergo rapid or
extreme fluctuation, otherwise temporary disruption of the
effectiveness of the bed may result. The flow velocity can
drop suddenly without affecting the bed, however sudden
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increases in velocity, if large enough, may disrupt the bed to
allow particles, such as white blood cells, to exit the fluid
chamber 22. -
The controller 40 increases flow into the fluid chamber
22 while maintaining the saturated fluidized bed. The
controller 40 may perform this flow rate increase by gradually
increasing flow in a continuous fashion until a final flow
rate is achieved. In a preferred embodiment, including the
fluid chamber 22 shown in Fig. 2, the controller 40 increases
the flow rate into the fluid chamber 22 by a ratcheting
process.
The controller 40 maintains the bed and increases the
flow rate in the ratcheting process by initially reducing the
rotational speed of the rotor 12. However, the rotational
speed of the rotor 12 is not reduced to a level that allows
red blood cells in outer layer 60 to spill over dam 50,
otherwise a significant number of both red blood cells and
white blood cells will flow into the fluid chamber 22. In an
embodiment of the invention, the controller 40 lowers the
rotational speed of the rotor 12 so that a centrifugal force
at dam 50 remains above 8506. Then, the controller ca7_culates
a value for K = Qi/Ni2, where K is a constant, Q is the
i
current flow rate into the fluid chamber 22, and Ni is the
current rotational speed of the centrifuge rotor 12.
After the controller 40 reduces the rotational speed of
the rotor 12 and calculates K, the controller 40
simultaneously increases both the flow rate into the fluid
chamber 22 and the rotational speed of the rotor 12. Thus, an
increasing centrifugal force in the fluid chamber 22
counteracts with increasing fluid flow forces in the fluid
chamber 22 to maintain the saturated fluidized particle bed as
a barrier to other particles. The new flow rate into the
chamber, Q, and the new rotational speed N satisfy the
equation: Q/N2 - K. Therefore, Q/N2 equals Q /N 2.
i i
To increase the flow rate even further, the controller 40
may continue to simultaneously increase both flow rate and
rotational speed_ Also, if necessary, the.controller 40 may
CA 02215986 2006-O1-10
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repeat the ratcheting process to further increase flow rate. ,
This is~accomplished by repeating the steps of reducing.
rotational speed and simultaneously increasing both flow rate
and rotational speed.
In addition, the controller 40 preferably returns the
saturated fluidized particle bed to its original state if a
pause in fluid flow to the fluid chamber 22 causes the bed to
collapse. The controller 40 may constantly monitor both flow
rate, Q, and rotational speed, N, to calculate a value for K =
Q/N2. If the flow into the fluid chamber 22 momentarily
pauses to collapse the saturated fluidized bed, the particles
that made up the bed temporarily remain in the fluid chamber
22. When fluid flow to the fluid chamber 22 is reinitiated,
the controller 40 controls both the flow rate, Q, and
rotational speed, N, so that these parameters satisfy the K =
Q/N2 relationship .existing immediately before fluid flow to
the fluid chamber 22 was interrupted. This automatically
returns the particles of the collapsed bed back into a
saturated fluidized bed form.
After a bed collapses the saturated fluidized particle
bed may recover in many other different ways. For example,
after a pause in fluid flow the flow rate may be increased in
a stepwise or gradual fashion to provide bed recovery.
Fig. 10 illustrates another embodiment of the invention.
In this embodiment a fluid chamber 522 is mounted on a _.
centrifuge rotor 512. The rotor 512 includes an outer bowl
521 and a clip 523 for holding a separation chamber 546 formed
in an elongated flexible tubular or belt shape from plastic
material. The device depicted in Fig. 10 separates particles,
in particular blood components, within the separation chamber
546 by generating centrifugal force. For further details
concerning the configuration and operation of this device,
refer to U.S. Patent 5,362,291 to Williamson, IV, U:S. Patent
5,360,542 to Brown et al., and U.S. Patent 5,078,671 to
Dennehev et al.
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The fluid chamber 522 is mounted or held on the
centrifuge rotor 512 with an outlet 532 facing substantially
toward an axis of rotation 513 of the centrifuge rotor 512.
Inflow tubing 542 supplies liquid and particles to the fluid
chamber 522 after the particles undergo an initial separation
within a portion of the separation chamber 546. In a similar
fashion, outflow tubing 538 conveys substances from the fluid
chamber 522 to another portion of the separation chamber 546.
In using the embodiment shown in Fig. lo, particles
carried by a liquid are separated in the separation. chamber
546 according to density-and size differences of the
particles. After being initially separated, the liquid and
particles, for example plasma carrying platelets and white
blood cells, flow into the fluid chamber 522. Within fluid
chamber 522, a saturated fluidized particle bed forms to
further separate particular particles from the flowing liquid.
Yet another embodiment of the invention is illustrated in
Figs. 11 and 12. This embodiment includes both a separation
chamber 646 shaped as a container and a collection container
627. Holders 629, 631 hold separation chamber 646 and
container 627, respectively, on a centrifuge rotor 612. Flow
lines 635, 638 provide fluid flow to and from the separation
chamber 646 and container 627 as indicated by the arrows
labelled with the symbol "E" shown in Fig. 11. Further
components include a restraining collar 637 and container _.
holder clamp assemblies 639. Particles are separated
according to density and size differences within the
separation chamber 646 in response to centrifugal force. For .
further details of how this device is configured and operates,
refer to Baxter Heathcare Corporation's CS-3000~ Plus Blood
Separator Operator's Manual (7-19-3-136).
As depicted in Figs. 11 and 12, a holder 624 holds a
fluid chamber 622 on the rotor 612. Inflow tubing 642 conveys
liquid and particles initially separated in separation chamber
646 into the fluid chamber 622. In addition, an outflow
tubing 638 fluidly couples the fluid chamber 622 to the
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collection container 627. With this configuration, a
saturated fluidized particle bed may be formed in the f-luid
- chamber 622, as explained with respect to the above
embodiments, to filter particles.
- Fig. 13 illustrates a further embodiment of the
invention. This embodiment includes a separation chamber 746,
preferably formed from rigid transparent plastic conduit,
capable of being inserted into a centrifuge. An inlet 741
allows whole blood to flow into a first stage 743 of the
separation chamber 746, so that red blood cells may be removed
from a conduit 745, while platelet rich plasma flows through
conduit 742. In addition, a conduit 747 and outlet 749 enable
removal of platelet poor plasma and platelets, respectively,
in a second stage 751. For further details regarding the
structural configuration and operation of this embodiment,
refer to the Brief Operating Instructions of the Fresenius MT
AS 104 blood cell separator (4/6.90(OP)), the disclosure of
which is incorporated herein by reference.
As shown in Fig. 13, a fluid chamber 722 may be coupled
to the separation chamber 746. A saturated fluidized particle
bed may be formed in the fluid chamber 722 to separate
particles after initial particle separation in the separation
chamber 746, in the manner described above.
In an alternate embodiment (not shown), the fluid chamber
722 may be coupled to outlet 749 of second stage 751. This
alternate embodiment would allow for particles to be separated
in a manner similar to the particle separation described for
the embodiment shown in Figs. 1-5.
Fig. 14 illustrates yet another embodiment of the
invention. As shown, a separation chamber 846 and a first
fluid chamber 822a are provided on a centrifuge rotor 812 in a
manner similar to that of the embodiment of Figs. 1-5. In
addition, an outflow tubing 838 fluidly couples an outlet 832a
of the fluid chamber 822a to an inlet 828b of an auxiliary
fluid chamber 822b. Mounting brackets (holders) 824a, 824b
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maintain the fluid chamber and the auxiliary chamber 822a,
822b, respectively, at substantially the same radial distance
from a rotation axis of the centrifuge rotor 812. '
In using the embodiment shown in Fig. 14, centrifuge
rotor 812 rotates to initially separate particles within '
separation chamber 846 according to density and/or
sedimentation velocity. Liquid carries separated particles
into the first fluid chamber 822a where particles further
separate after formation of a saturated fluidized bed of
particles or an elutriation field. Thereafter, the separated
particles and liquid flow through tubing 838 into auxiliary
fluid chamber 822b where particles are further separated by
either a saturated fluidized bed o-f particles or an
elutriation field. Thus, particles separate within chambers
822a, 822b by forming a saturated fluidized particle bed in
one of the chambers 822a, 822b and an elutriation boundary in
another of the chambers 822a, 822b, or, in the alternative, by
forming either a saturated fluidized particle bed or an
elutriation boundary in both of the chambers 822a, 822b.
Optionally the chambers 822a, 822b may have different
dimensions, such as differing volumes, lengths or maximum
diameters. For example, fluid chamber 822a may have a greater
volume than that of auxiliary chamber 822b. These different
dimensions allow for two different particle separations to
take place within each of the chambers 822a, 822b.
The embodiment of Fig. 14 allows for multiple particle
separations to take place simultaneously. Additionally,
different types of particles may be harvested in one single
procedure. Of course, one or more further fluid chambers may
be added without departing from the scope of the invention.
Furthermore, both of the chambers 822a and 822b may be
cylindrical.
It will be apparent to those skilled in the art that
various modifications and variations can be made to the
structure and methodology of the present invention without
departing from the scope or spirit of the invention.
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The fluid chambers 122, 222, 322, 422, 1022, or 1022'
depicted in the embodiments of Figs. 6-9, 16 and 17, may be
- substituted for the fluid chambers 22, 522, 622, 722, 822a or
822b. Further, the invention can be modified by including
- supplemental fluid chambers or separation chambers. Although
the invention has been described as having a separation
chamber to initially separate particles from the liquid, the
inventior~ can be practiced without this initial separation
taking place.
Although the controller 40 is described above as
controlling rotor speed and flow rate, the controller 40 may
also regulate other parameters. For example the controller
may control the density or some other characteristic of the
liquid used to carry particles into the fluid chamber.
In addition, while the invention is described herein in
connection with blood component separation, the invention in
its broadest sense is not so limited. The invention is
applicable to other medical and non-medical uses.
The particles used to form the saturated fluidized bed in
the fluid chamber can differ from the particles within the
fluid passing through the fluid chamber for filtration.
Additionally, it is possible to initially form the saturated
fluidized bed with an extremely high concentration of
platelets having very few white blood cells.
Further, the fluid chamber of the invention may be used
in a separation process involving elutriation or any other
particle separation means without departing from the scope of
the invention.
In accordance with the invention there is also provided
means for altering sedimentation velocity of particles used to
form the saturated bed. Figs. 18-20 illustrate embodiments of
the invention including examples of such means. As
illustrated in Fig. 18, a portion of a tubing set 1100
preferably includes a primary container 1102 for containing a
primary substance including liquid and particles to be
separated.
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In a preferred embodiment of the invention, the primary
substance in primary container 1102 is a peripheral cell
collection including, for example, plasma, red blood cells, -
stem cells, T cells and optionally tumor cells. As mentioned
above, devices for purifying blood to obtain a peripheral cell
collection are known in the art. As described in more detail
below, particular particles, such as red blood cells, in the
primary substance are used to form a saturated fluidized
particle bed in a fluid chamber 1122 or 1122' shown
respectively in Figs 19 and 20. Fluid chambers 1122 and 1122'
are preferably constructed in a manner similar to one of the
fluid chambers 22, 122, 222, 322, 422, 522, 622, 722, 822a,
822b, 1022, or 1022' described above.
As shown in Fig. 18, the tubing set 1100 also preferably
includes a series of additive containers 1104 containing
additives for altering sedimentation velocity of the particles
in the primary substance forming the saturated fluidized bed
in the fluid chamber 1122, 1122'. A series of outflow tubes
1112 and 1114 respectively couple the primary container 1102
and each of the additive containers 1104 to a supply line 1121
of the tubing set 1100 so that the primary substance and
additives flowing in the outflow tubes 1112 and 1114 mix in
the supply line 1121. As shown respectively in Figs. 19 and
20, the supply line 1121 is coupled to an inlet 1140, 1140' of
the fluid chamber 1222, 1222' (respectively).
A series of pumps 1124 and 1126, shown schematically a.n
Fig. 18, regulate flow in the outflow tubes 1112 and 1114 from
the primary container 1102 and additive containers 1104 to the
supply line 1121. Preferably, the pumps 1124 and 1126 are
peristaltic pumps connected to a controller 1106, similar to
the controller 40 described above in connection with Fig. 3.
Controller 1106 meters the amount of primary substance and
additives flowing and mixing in the supply line 1121.
As will be recognized by those skilled in the art, other
types of flow regulating devices, such as, for example, valves
or impeller pumps, could be used in place of the peristaltic
pumps 1124 and 1126. In its broadest sense., the invention
~
CA 02215986 1997-10-03
' , ~ "
;, , '~,; ,'
- ' ; ,
- 43 - ' 7 -,., " "' , ..
a
does not require all of the above-described structure. For
example, the additive substances could be introduced directly
into the primary container 1102 without the use additive
containers 1104, pumps 1126, and outflow tubes 1114. When the
particles forming the saturated fluidized bed are magnetic, a
variable magnetic field adjacent to the fluid chamber 1122,
1122' could alter sedimentation velocity of the bed forming
particles.
With the structure described above, it is possible to
alter the sedimentation velocity of particles forming a
saturated fluidized bed in chamber 1122, 1122'. By changing
sedimentation velocity, as described below, filtration
characteristics of the saturated fluidized bed may be altered.
In use, one or more of the additive substances in
containers 1104 are chosen because of their ability to change
the sedimentation velocity of the bed forming particles to a
sedimentation velocity greater than that of first particles in
the primary substance and less than that of second particles
in the primary substance. Controller 1106 regulates pumps
1126 to precisely control the sedimentation velocity of the
particles in the saturated fluidized bed. After the
sedimentation velocity of the bed forming particles is
properly adjusted, the bed allows certain particles to pass
through the fluid chamber 1122, 1122' while other particles
are retained by the bed in fluid chamber 1122, 1122'.
When sedimentation velocity of the bed forming particles
is increased, the saturated fluidized bed retains particles in
the fluid chamber 1122 or 1122' that would normally pass
through the bed if the sedimentation velocity of the bed
forming particles was not altered. Conversely, when
sedimentation velocity of the bed forming particles is
decreased, the saturated fluidized bed allows particles to
pass through the bed that would normally be retained in the
fluid chamber 1122 or 1122' if the sedimentation velocity of
the bed forming particles was not altered.
A~FMOFD SHEFT
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In a preferred embodiment of the invention, red blood
cells may be used to form the saturated fluidized bed, and one
of the additive containers 1104 may contain an additive
substance such as water or saline having a weak concentration
of salt. For example, the additive substance in one of the
additive containers 1104 may be a saline solution having about
0.4o sodium chloride by weight. Such additive substances are
hypotonic as compared to red blood cells_ Another of the
additive containers 1104, preferably contains an additive
substance, such as saline having a strong concentration of
salt, which is hypertonic as compared to red blood cells. For
example, the hypertonic additive solution in one of the
additive containers 1104 may be a saline solution having about
7o sodium chloride by weight.
The hypotonic or hypotonic additive substances may be
many different types of solutions containing a predetermined
concentration of a particular solute. For example, these
substances may include albumin as a solute.
As described in more detail below, hypotonic additive
solutions tend to osmotically increase the size of red blood
cells. Conversely, hypertonic solutions tend to osmotically
decrease red blood cell size. Particle sedimentation
velocity, as previously discussed, is directly related to the
size of the particle according to Stokes law. Therefore, an
increase in particle size increases sedimentation velocity,
while a decrease in particle size decreases sedimentation
velocity.
Another of the additive containers 1104 preferably holds
a solution containing albumin or sucrose. Such solutions
change the density and/or viscosity of liquid, such as plasma,
flowing from the primary container 1102. As mentioned above,
sedimentation velocity of a particle is also related to the
density and viscosity of a fluid according to Stokes law.
Therefore, by adding density/viscosity altering components
into supply line 1121, sedimentation velocity of the saturated
fluidized bed particles may be altered.
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At least one of the additive containers 1104 preferably
contains a substance that links a number of the saturated
fluidized bed forming particles in the primary solution
together to form a series of saturated bed forming particle
groups having a size larger than that of the individual
particles. Because the particles groups are larger than the
individual particles, sedimentation velocity of the saturated
bed forming particle groups is greater than that of the
individual particles according to Stokes law. In a preferred
embodiment, such a particle group forming substance contains
macromolecules of dextran or fibrogen. These substances cause
red blood cells to rouleau -that is the red blood cells link
together and form separate red blood cell groups.
In an embodiment of the invention shown in Fig. 19, the
supply line 1121 is coupled to the inlet 1140 of fluid chamber
1122. The fluid chamber 1122 is mounted to a centrifuge rotor
1134 of a centrifuge device 1136 preferably including a motor,
shaft, arm, holder, pump, and controller 1106 respectively
similar or identical to the motor 16, shaft 18, arm 19, holder
24, pump 36, and controller 4G shown in Figs. 1 and 3.
In an alternate embodiment of the invention shown in Fig.
20, the supply line 1121 is coupled to an inlet 1140' of fluid
chamber 1122'. The fluid chamber 1122' is mounted to a
centrifuge rotor 1134' of a centrifuge device 1136' configured
like the centrifuge device 1136 shown in Fig. 19.
In accordance with the invention there is also provided
means for separating particles from liquid after the particles
and liquid flow from the fluid chamber. As illustrated in
Fig. 19, intermediate tubing 1138 couples an outlet 1142 of
fluid chamber 1122 to an inlet 1144 of a supplemental chamber
1146. Supplemental chamber 1146 is removably mounted on the
centrifuge rotor 1134.
As shown in Fig. 19, the fluid chamber 1122 and
supplemental chamber 1146 are spaced at approximately the same
distance from an axis of rotation of rotor 1134. A maximum
cross-sectional area of the supplemental chamber 1146 is
preferably larger than that of the fluid chamber 1122 so that
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the supplemental chamber 1146 retains particles without
forming a saturated fluidized particle bed. In an alternate
embodiment (not shown), the supplemental chamber 1146 is
further than fluid chamber 1122 from an axis of rotation of
rotor 1134. During rotation of rotor 1134, this spacing
allows particles in the supplemental chamber 1146 to encounter
a stronger centrifugal force than particles in fluid chamber
1122, thus allowing the supplemental chamber 1146 to have a
maximum cross secti-onal area similar to that of fluid chamber
1122.
As described in more detail below, by controlling flow
rate and rotation of rotor 1134, centrifugal force in the
supplemental chamber 1146 causes particles passing from fluid
chamber 1122 to be retained in chamber 1146 while permitting
liquid to pass through an outlet 1148 and into an outlet line
1150.
As shown in Fig. 19, the supplemental chamber 1146 is
preferably larger than the fluid chamber 1122, permitting the
supplemental chamber 1146 to retain a substantial number of
particles. Preferably, an interior of the supplemental
chamber 1146 is shaped like that of the fluid chamber 1122.
In an alternative embodiment shown in Fig. 20,
intermediate tubing 1138' couples an outlet 1142' of fluid
chamber 1122' to an inlet 1152 of a separation channel 1154
mounted to centrifuge rotor 1134'. Adjacent to an outer
portion of the centrifuge rotor 1134', the separation channel
1154 has a collection well 1156 for collecting particles
flowing into the separation channel 1154. Rotation of
centrifuge rotor 1134' sediments particles into the collection
well 1156 while slower sedimenting liquid and possibly some
slower sedimenting particles remain above a top boundary 1158 _
of the collection well.
As shown in Fig. 20, collection well 1156 has a particle
concentrate outlet 1164 connected to a particle concentrate
line 1160. The particle concentrate line 1160 removes
particles retained in the collection well 1156. A liquid
outlet 1166 is provided above top boundary.l158 and is
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connected to liquid outlet line 1162. The liquid outlet line
1162 removes liquid above the top boundary 1158. In addition,
the liquid outlet line 1162 may remove slower sedimenting
particles above the top boundary 1158.
- Preferably, the particle collection well 1156, particle
concentrate outlet 1164 and liquid outlet 1166 are located at
or adjacent to one end of the separation channel 1154, and the
inlet 1152 is located at or adjacent to an opposite end of the
separation channel 1154. This spacing ensures ample time for
separation of particles from liquid, collection of a
substantial number of particles in the collection well 1156,
and corresponding removal of a substantial number of particles
through the concentrate line 1160.
Methods for separating T cells, tumor cells, stem cells,
and/or plasma from a peripheral cell collection are discussed
below with reference to Figs. 18-20. Although the invention
is described in connection with separating these substances
from a peripheral cell collection, it should be understood
that the invention in its broadest sense is not so limited.
For example, the invention may be readily practiced to
separate substances in a bone marrow harvest collection or an
umbilical cord cell collection harvested following birth. In
addition, the method of the invention in its broadest sense
could be practiced with structure different from that
described in connection the embodiments shown in Figs, 18-20.
After a donation procedure in which a peripheral cell
collection is obtained, the peripheral cell collection is
placed in the primary container 1102. A substantial portion
of the peripheral cell collection includes plasma, red blood
cells, stem cells, and T cells. If the donor's blood included
tumor cells, these cells are also present in the peripheral
cell collection.
Ultimately, the red blood cells from the peripheral cell
collection will be used to form a saturated fluidized bed in
the fluid chamber 1122, 1122'. If the number of red blood
cells in the peripheral cell collection is insufficient to
form the saturated bed, additional red blood cells are
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4g
preferably added to the primary container 1102 so that the
number of red blood cells exceeds the number of stem cells, T
cells, and any tumor cells in the primary container 1102.
At the beginning of a particle separation procedure, the
pump 1124 associated with container 1102 is activated to
convey the peripheral cell collection from the primary
container 1102 to the supply line 1121. In addition, one or
more of pumps 1126 associated with the additive substances
from additive containers 1104 are used to supply additives to
supply line 1121. In the supply line 1121, the peripheral
cell collection and one or more of the additive substances mix
to increase or decrease the sedimentation velocity of the red
blood cells.
When hypotonic solution is added to supply line 1121, the
red blood cells osmotically increase in size thereby
increasing the sedimentation velocity of the red blood cells.
Conversely, when the hypertonic solution is added to supply
line 1121, the red blood cells decrease in size thereby
reducing the sedimentation velocity of the red blood cells.
When saline is used as the hypotonic or hypertonic solution,
the mixture of the peripheral cell collection and additive
substances in supply line 1121 may include about 0.6% to about
5o sodium chloride by weight. Preferably, the amount of
sodium chloride or other solutes in the supply line 1121 is
sufficient to prevent hemolysis (bursting) of the red blood
cells if the red blood cells increase in size. For example,
when saline is used, the amount of sodium chloride in 'the
supply line 1121 is preferably above about 0.4o by weight.
When a solution containing albumin or sucrose is added to
supply line 1121, the solution increases the density and/or
viscosity of plasma in the supply line 1121, thereby reducing
sedimentation velocity of the red blood cells. When a medium
including macromolecules of dextran or fibrogen are added to
the supply line 1121, the medium causes red blood cells in the
supply line 1121 to rouleau, thereby increasing the
sedimentation velocity of the red blood cells.
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The controller 1106 activates one or more of the pumps
1126 depending on the sedimentation velocity of the red blood
- cells and the sedimentation velocities of cells that will be
separated. For example, to separate faster sedimenting stem
cells from slower sedimenting T cells, additive introduction
is controlled so that the sedimentation velocity of the red
blood cells falls between the sedimentation velocity of the
stem cells and the sedimentation velocity of the T cells.
The peripheral cell collection and additive substances
flow through the supply line 1121 into the inlet 1140, 1140'
of the fluid chamber 1122, 1122'. The controller 1106
controls the rotational speed of the rotor 1136, 1136' and the
rate of flow into the fluid chamber 1122, 1122' to establish a
saturated fluidized bed of red blood cells in the fluid
chamber 1122, 1122'.
The saturated fluidized bed of red blood cells behaves
like the earlier-described saturated fluidized bed of
platelets. Plasma, additive substances, and slower
sedimenting T cells pass through the saturated fluidized red
blood cell bed and outlet 1142, 1142', while the bed retains
faster sedimenting stem cells in the fluid chamber 1122,
1122'. As red blood cells continue to flow into the fluid
chamber 1122, 1122' and enter the saturated fluidized bed, red
blood cells flow from the outlet 1142, 1142'. Because the
fluidized bed is saturated, the rate at which red blood cells
enter the inlet 1140, 1140' equals the rate at which red blood
cells pass through the outlet 1142, 1142'.
As the saturated fluidized red blood cell bed continues
to filter faster sedimenting particles, the pump 1124 and one
or more of additive pumps 1126 continue to mix the peripheral
cell collection and additives in the supply line 1121. This
ensures that red blood cells entering the saturated fluidized
bed and red blood cells in the bed have a sedimentation
velocity between that of the first and second particles being
separated.
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5p _
In the embodiment of Fig. 19, plasma, additive
substances, T cells, and the portion of red blood cells
leaving the saturated fluidized bed flow through the outlet
1142 and intermediate tubing 1138 into the inlet 1144 of
supplemental chamber 1146. In the supplemental chamber 1146,
centrifugal force caused by rotation of the rotor 1134 retains
the T cells and red blood cells. Meanwhile, the plasma and
additive substances flow from the outlet 1148 into the outlet
line 1150. This separates the T cells and red blood cells
from the plasma and additive substances.
The stem cells and red blood cells in the fluid chamber
1122 and the T cells and red blood cells in the supplemental
chamber 1146 may retain a residue of some of the additive
substances, such as albumin, sucrose, dextran, or fibrogen.
To wash this residue from these cells, one of pumps 1126
preferably pumps a washing medium such as saline from a
corresponding additive container 1104 through the fluid
chamber 1122 and supplemental fluid chamber 1146 before
completion of the separation procedure. If necessary,
rotational speed of the rotor 1134 may be altered to retain
particles in the fluid chamber 1122 and supplemental fluid
chamber 1146 during washing.
When all of the peripheral cell collection flows from
primary container 1102, the controller 1106 terminates
rotation of the centrifuge rotor 1134. To remove the stem
cells and red blood cells from the fluid chamber 1222, a
procedurist releases the fluid chamber 1122 from the
centrifuge rotor 1134 and separates the supply line 1121 or
intermediate tubing 1138 from the fluid chamber 1122_ In a
similar fashion, the procedurist removes T cells and red blood
cells retained in the supplemental fluid chamber1146.
In the embodiment of Fig. 20, plasma, additive
substances, T cells, and the portion of red blood cells
leaving the saturated fluidized bed flow through the outlet
1142' and intermediate tubing 1138' into the inlet 1152 of
separation channel 1154. Centrifugal force generated by the
rotation of centrifuge rotor 1134' sediments the T cel7_s and
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red blood cells in the collection well 1156, while plasma and
additive substances remain above the top boundary 1158 of the
' collection well 1156. As the T cells and red blood cells
collect in the collection well 1156, the particle concentrate
- line 1160 removes them through outlet 1164. Simultaneously,
the liquid outlet line 1162 removes plasma and additive
substances through the liquid outlet 1166.
When the primary container 1102 empties, the controller
1106 terminates rotation of the rotor 1136'. A procedurist
then removes the stem cells and red blood cells from the fluid
chamber 1222' by releasing the fluid chamber 1122' from the
centrifuge rotor 1134' and separating the supply line 1121' or
intermediate tubing 1138' from the fluid chamber 1122'.
To separate faster sedimenting tumor cells from slower
sedimenting stem cells, one or more of the pumps 1126 add
additive substances from one or more of the additive
containers 1104 into the supply line 1121. The additive
substances adjust the sedimentation velocity of the red blood
cells to a sedimentation velocity between the sedimentation
velocity of tumor cells and the sedimentation velocity of stem
cells.
As described above, a saturated fluidized bed of red
blood cells forms in the fluid chamber 1122, 1122'. Because
the tumor cells have a sedimentation velocity greater than
that of the red blood cells, the saturated fluidized red blood
cell bed retains tumor cells in the fluid chamber 1122, 1122'.
Stem cells, which now have a sedimentation velocity less than
that of the red blood cells, flow from the outlet 1142, 1142'
of fluid chamber 1122, 1122' along with the portion of red
blood cells leaving the bed.
In the supplemental chamber 1146 or the separation
channel 1154, stem cells and red blood cells separate from the
plasma and additive substances in the same way the T cells and
red blood cells separate from the plasma and additive
substances in the method described above. When the embodiment
of Fig. 19 is used, washing medium, such as saline from one of
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additive containers 1104, preferably washes additive residue
from the particles in fluid chamber 1122, and in supplemental
fluid chamber 1146, as described above.
When the primary container 1102 is empty, a procedurist
removes tumor cells and red blood cells from the fluid chamber
1122 or 1122'. Stem cells and the red blood cells are removed
from the supplemental chamber 1146 shown in Fig. 19 or from
the particle concentrate outlet shown in Fig. 20.
The invention may be used to separate slower sedimenting
tumor cells from faster sedimenting stem cells by retaining
stem cells in the saturated fluidized red blood cell bed,
while allowing tumor cells to pass through the bed. The
invention, in its broadest sense, may also be used to separate
many different types of particles from one another. Thus, it
should be understood that the invention is not limited to the
examples discussed in this specification. Rather the
invention is intended to cover modifications and variations
provided they come within the scope of the following claims.
AMEidDED SNEEI
