U.S. patent application number 12/500077 was filed with the patent office on 2010-07-08 for system and method for removing a cryporotectant from a liquid.
Invention is credited to Alan Robert Brunsman.
Application Number | 20100170848 12/500077 |
Document ID | / |
Family ID | 42311026 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100170848 |
Kind Code |
A1 |
Brunsman; Alan Robert |
July 8, 2010 |
System and Method for Removing a Cryporotectant from a Liquid
Abstract
A system for removing cryoprotectant from a
cryoprotectant-containing liquid stored a container comprises a
cryoprotectant removal device that receives the
cryoprotectant-containing liquid and a cryoprotectant-free
dialysate liquid and that is operable to transfer cryoprotectant to
the dialysate liquid. A differential conductivity device is
arranged to continuously measure the difference in conductivity
between dialysate liquid entering the device and dialysate liquid
that has received cryoprotectant transferred by the dialyzer
discharged from the device. A controller is operable to control the
flow of the liquids through the device in response to the measured
difference in conductivity, and particularly to stop the flow of
the cryoprotectant-containing liquid when the measured differential
conductivity indicates that the cryoprotectant has been
substantially removed from the liquid.
Inventors: |
Brunsman; Alan Robert;
(Yellow Springs, OH) |
Correspondence
Address: |
MAGINOT, MOORE & BECK, LLP;CHASE TOWER
111 MONUMENT CIRCLE, SUITE 3250
INDIANAPOLIS
IN
46204
US
|
Family ID: |
42311026 |
Appl. No.: |
12/500077 |
Filed: |
July 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61079282 |
Jul 9, 2008 |
|
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Current U.S.
Class: |
210/637 ;
210/96.2 |
Current CPC
Class: |
A61M 1/1682 20140204;
A61M 1/3669 20130101; B01D 61/32 20130101; A61M 1/0281
20130101 |
Class at
Publication: |
210/637 ;
210/96.2 |
International
Class: |
B01D 61/34 20060101
B01D061/34 |
Claims
1. A system for removing cryoprotectant from a
cryoprotectant-containing liquid stored a container comprising: a
dialyzer having a first inlet to receive the
cryoprotectant-containing liquid, a first outlet for discharge of
the cryoprotectant-liquid, a second inlet to receive a
cryoprotectant-free dialysate liquid and a second outlet for
discharge of the cryoprotectant-free liquid, said dialyzer forming
a diffusion gradient between said cryoprotectant-containing liquid
and the cryoprotectant-free liquid; a source of cryoprotectant-free
dialysate liquid capable of receiving cryoprotectant transferred
through said diffusion gradient within said dialyzer; an outlet
fluid line connected to said first inlet and connectable to an
outlet of the container; an inlet fluid line connected to said
first outlet and connectable to an inlet of the container, said
outlet and inlet fluid lines forming a first fluid circuit between
the container of the cryoprotectant-containing liquid and said
dialyzer; a second outlet fluid line connected between said source
of dialysate liquid and said second inlet; a discharge fluid line
connected to said second outlet and connectable to a waste
container, said second outlet fluid line and said discharge fluid
line forming a second fluid circuit between said source of
dialysate liquid and said dialyzer; a first pump disposed in said
first fluid circuit for controlling the flow of the
cryoprotectant-containing liquid through said first fluid circuit;
a second pump disposed in said second fluid circuit for controlling
the flow of the dialysate liquid through said second fluid circuit;
a differential conductivity device disposed between said second
outlet fluid line and said discharge fluid line and operable to
measure the difference in conductivity between dialysate liquid
flowing through said second outlet fluid line and dialysate liquid
that has received cryoprotectant transferred by said dialyzer
flowing through said discharge fluid line; and a controller for
controlling the operation of said first and/or second pump in
response to the measured difference in conductivity.
2. The system for removing cryoprotectant according to claim 1,
further comprising: a first valve disposed in said first outlet
fluid line, said first valve openable to permit and closable to
prevent flow of cryoprotectant-containing liquid from the container
to said dialyzer; and a second valve disposed in said second outlet
fluid line, said second valve openable to permit and closable to
prevent flow of dialysate liquid from said source to said dialyzer,
wherein said controller is configured to open or close said first
and/or second valve in response to the measured difference in
conductivity.
3. The system for removing cryoprotectant according to claim 1,
further comprising: a first temperature sensor adjacent said
differential conductivity device and arranged to measure the
temperature of liquid flowing through said second outlet fluid
line; and a second temperature sensor adjacent said differential
conductivity device and arranged to measure the temperature of
liquid flowing through said discharge fluid line, wherein said
controller is further configured to adjust the measured difference
in conductivity as a function of the temperature sensed by said
first and second temperature sensors.
4. The system for removing cryoprotectant according to claim 1,
further comprising: a source of a priming solution for preparing
said dialyzer; a first priming outlet fluid line connected between
said source of priming solution and said first inlet of said
dialyzer; and a second priming outlet fluid line connected between
said source of priming solution and said second inlet of said
dialyzer.
5. The system for removing cryoprotectant according to claim 4,
wherein: said first priming outlet fluid line intersects said first
fluid circuit so that said first pump is operable to flow said
priming solution through said dialyzer; and said second priming
outlet fluid line intersects said second fluid circuit so that said
second pump is operable to flow said priming solution through said
dialyzer and so that said priming solution flows through said
differential conductivity device.
6. The system for removing cryoprotectant according to claim 5,
further comprising: a third valve disposed in said first priming
outlet fluid line, said third valve openable to permit and closable
to prevent flow of priming solution from said source of priming
solution to said dialyzer; and a fourth valve disposed in said
second priming outlet fluid line, said fourth valve openable to
permit and closable to prevent flow of priming solution from said
source of priming solution to said dialyzer, wherein said
controller is configured to open or close said third and/or fourth
valve in response to the measured difference in conductivity.
7. The system for removing cryoprotectant according to claim 1,
further comprising: a conductivity cell disposed in said inlet
fluid line adjacent said dialyzer, said conductivity cell arranged
to measure the conductivity of liquid flowing through said inlet
fluid line; a waste fluid line intersecting said inlet fluid line
between said conductivity cell and the inlet to the container of
the cryoprotectant-containing liquid, said waste fluid line
connectable to a waste container; and a fifth valve disposed in
said waste fluid line between said inlet fluid line and the waste
container, said fifth valve openable to permit and closable to
prevent flow of liquid from said dialyzer to the waste container,
wherein said controller is configured to open or close said fifth
valve in response to the measured conductivity.
8. The system for removing cryoprotectant according to claim 7,
further comprising: a third temperature sensor adjacent said
conductivity cell and arranged to measure the temperature of liquid
flowing through said inlet fluid line, wherein said controller is
further configured to adjust the measured conductivity as a
function of the temperature sensed by said third temperature
sensor.
9. A method for removing cryoprotectant from a liquid comprising
the steps of: passing a cryoprotectant-containing liquid and a
cryoprotectant-free liquid through a cryoprotectant removal device
configured to transfer cryoprotectant from the
cryoprotectant-containing liquid to the cryoprotectant-free liquid;
measuring the differential conductivity between cryoprotectant-free
liquid entering the device and cryoprotectant-free liquid
discharged from the device after receiving cryoprotectant
transferred within the device; and controlling the flow of the
cryoprotectant-containing liquid and/or the cryoprotectant-free
liquid through the device in response to the measured differential
conductivity.
10. The method for removing cryoprotectant of claim 9, wherein the
step of controlling the flow comprises continuously comparing the
measured differential conductivity value to a predetermined
threshold value indicative that the liquid discharged from the
device is substantially free cryoprotectant.
12. The method for removing cryoprotectant of claim 9, further
comprising the steps of: passing a priming solution through the
device prior to operating the device to remove cryoprotectant; upon
completion of the priming step, flowing cryoprotectant-containing
liquid through the device; continuously measuring the conductivity
of liquid discharged from the device; and diverting the liquid
discharged from the device to a waste container until the measured
conductivity reaches a predetermined threshold value indicative
that only cryoprotectant-containing liquid is flowing through the
device.
13. The method for removing cryoprotectant of claim 9, wherein the
measuring step includes measuring the temperature of the
cryoprotectant-free liquid entering the device and liquid
discharged from the device and adjusting the differential
conductivity as a function of the measured temperatures.
Description
REFERENCE TO RELATED APPLICATION
[0001] The present application claims priority to co-pending
provisional application No. 61/079,282, filed on Jul. 9, 2008, in
the name of the present inventor, and entitled "System for
Detecting the Presence of a Substance in a Fluid." The disclosure
of this application No. 61/079,282 is incorporated herein by
reference.
BACKGROUND
[0002] The present disclosure concerns systems and methods for
detecting the presence of substances in a liquid, such as blood and
other bodily liquids. One application of the system and method
disclosed herein is to detect the presence of preservation
substances, such as DMSO, in a cryogenically-treated blood sample
that is being treated to remove the DMSO.
[0003] It is known to utilize various cryoprotectants, such as
dimethyl sulfoxide (DMSO), during cryopreservation cells. Use of a
cryoprotectant is essential to prevent cryoinjury to the cells,
such as from the formation of intracellular ice crystals during
freezing. Thus, in stem cell transplant treatments, for instance,
the stem cells are obtained and frozen, to be later thawed for
periodic treatments of a patient affected by cancer or other
diseases. In some prior treatments, the frozen-thawed stem cells
are injected into the patient, along with the cryoprotectant,
because there have been no effective ways to remove the
cryoprotectant without losing a significant amount of stems cells
or otherwise contaminating them. However, at room or body
temperature, certain cryoprotectants, such as DMSO, are known to be
toxic to cells as well as the patient. For instance, DMSO is known
to cause ill effects in patients, ranging from fever and nausea to
violent cramping. In some cases, the presences of cryoprotectant
may endanger the patient's life. The potentially dangerous effects
of cryoprotectants on the patient has tempered the desirability of
using frozen and banked cells or liquids of any type.
[0004] One common method for removal of cryoprotectant has been
mechanical removal, typically in the form of centrifugation
followed by resuspension in a media to remove the cryoprotectant by
dilution. However, the mechanical forces introduces during
centrifugation result in osmotic stress and cell clumping/lysing,
particular for fragile cells. Moreover, the generally open nature
of centrifugation may result in bacterial or viral contamination of
the cell preparation.
[0005] In order to address these problems a closed system has been
developed as disclosed in U.S. Pat. No. 6,869,758 (the '758
patent), assigned to the University of Kentucky Research
Foundation. The disclosure of the '758 patent is incorporated
herein by reference. The '758 patent discloses passing the
cryoprotectant-containing liquid through at least one semipermeable
hollow fiber membrane contained in a hollow module in a first
direction to contact the hollow fiber membrane on at least one
interior surface. Concurrently, a liquid which is substantially
free of cryoprotectant is passed through the hollow module in a
second direction (opposite the first direction) so that the
cryoprotectant-free liquid contacts the semipermeable hollow fiber
membrane on at least one exterior surface. A diffusion gradient is
thus created that transfers the cryoprotectant from the
cryoprotectant-containing liquid to the cryoprotectant-free liquid
for subsequent removal.
[0006] Thus, in the treatment of a frozen-thawed cell suspension
containing a cryoprotectant, the hollow module and semipermeable
hollow fiber membrane disclosed in the '758 patent can be connected
directly to the source of the suspension. In the case of
frozen-thawed blood, the device disclosed in the '758 patent can be
connected to the blood bag in a closed system. The system may
incorporate a series of pumps and valves to move the cell
suspension liquid and the cryoprotectant-free liquid through the
system. Details of one such system are shown in FIG. 2 and
described herein.
[0007] The system disclosed in the '758 patent provides a
completely closed system for the effective removal of
cryoprotectant from a liquid. Since the system relies upon
diffusion and the dialysis process, there is no damage to the
desired cells if the process is optimally performed. Moreover, the
process retains a significant quantity of the original
frozen-thawed liquid, again if optimally performed. In order to
achieve optimal performance, it is desirable that the
cryoprotectant removal process continue only for as long as
necessary to reduce the presence of cryoprotectant in the cell
suspension to a suitable level. While the closed system is less
harmful to the desired cells than the prior mechanical methods,
"over-treatment" of the cells can cause damage and reduce the
quantity of viable cells. On the other hand, "under treatment" does
not remove enough of the cryoprotectant, so that the damaging
effects of the cryoprotectant remain. Thus, there is a need for a
system and method for determining when the dialysis process is
complete.
SUMMARY
[0008] According to one aspect of the invention, a system is
provided for removing cryoprotectant from a
cryoprotectant-containing liquid stored a container that comprises
a cryoprotectant removal device that receives the
cryoprotectant-containing liquid and a cryoprotectant-free
dialysate liquid and that is operable to transfer cryoprotectant to
the dialysate liquid. A differential conductivity device is
arranged to continuously measure the difference in conductivity
between dialysate liquid entering the device and dialysate liquid
that has received cryoprotectant transferred by the dialyzer
discharged from the device. A controller is operable to control the
flow of the liquids through the device in response to the measured
difference in conductivity, and particularly to stop the flow of
the cryoprotectant-containing liquid when the measured differential
conductivity indicates that the cryoprotectant has been
substantially removed from the liquid.
[0009] In a further aspect, the cryoprotectant removing device is a
dialyzer having a first inlet to receive the
cryoprotectant-containing liquid, a first outlet for discharge of
the cryoprotectant-liquid, a second inlet to receive a
cryoprotectant-free dialysate liquid and a second outlet for
discharge of the cryoprotectant-free liquid, the dialyzer forming a
diffusion gradient between the cryoprotectant-containing liquid and
the cryoprotectant-free liquid. The system further comprises an
outlet fluid line connected to the first inlet and connectable to
an outlet of the container, and an inlet fluid line connected to
the first outlet and connectable to an inlet of the container, the
outlet and inlet fluid lines forming a first fluid circuit between
the container of the cryoprotectant-containing liquid and the
dialyzer. A second outlet fluid line is connected between the
source of dialysate liquid and the second inlet, and a discharge
fluid line is connected to the second outlet and connectable to a
waste container, the second outlet fluid line and the discharge
fluid line forming a second fluid circuit between the source of
dialysate liquid and the dialyzer.
[0010] A first pump is disposed in the first fluid circuit for
controlling the flow of the cryoprotectant-containing liquid
through the first fluid circuit, and a second pump is disposed in
the second fluid circuit for controlling the flow of the dialysate
liquid through the second fluid circuit. In one feature, the
differential conductivity device is disposed between the second
outlet fluid line and the discharge fluid line and is operable to
measure the difference in conductivity between dialysate liquid
flowing through the second outlet fluid line and dialysate liquid
that has received cryoprotectant transferred by the dialyzer
flowing through the discharge fluid line. The controller is
configured to control the operation of the first and/or second pump
in response to the measured difference in conductivity.
[0011] A method is provided for removing cryoprotectant from a
liquid which comprising the step of passing a
cryoprotectant-containing liquid and a cryoprotectant-free liquid
through a cryoprotectant removal device configured to transfer
cryoprotectant from the cryoprotectant-containing liquid to the
cryoprotectant-free liquid. In one feature, the method includes
measuring the differential conductivity between cryoprotectant-free
liquid entering the device and cryoprotectant-free liquid
discharged from the device after receiving cryoprotectant
transferred within the device. The method further contemplates
controlling the flow of the cryoprotectant-containing liquid and/or
the cryoprotectant-free liquid through the device in response to
the measured differential conductivity.
DESCRIPTION OF THE FIGURES
[0012] FIG. 1 is a side view of a conductivity cell for use with a
closed system for removing cryoprotectant from a liquid.
[0013] FIG. 2 is a diagram of a closed system for removing
cryoprotectant from a liquid, incorporating the conductivity cell
shown in FIG. 1, with the system in a first state for priming the
system.
[0014] FIG. 3 is a diagram of the closed system shown in FIG. 2,
with the system in a second state for commencing blood flow from a
blood bag through the system.
[0015] FIG. 4 is a diagram of the closed system shown in FIG. 2,
with the system in a third state for directing blood flow back to
the blood bag.
[0016] FIG. 5 is a diagram of the closed system shown in FIG. 2,
with the system in a subsequent state for directing flow of
dialysate through the system.
[0017] FIG. 6 is a diagram of the closed system shown in FIG. 2,
with the system in a fifth state for recovering blood cells
remaining in the system after the prior steps.
[0018] FIG. 7 is a diagram of the closed system shown in FIG. 2,
with the system in a further state for recovering additional blood
cells remaining in the system after the prior steps.
DESCRIPTION OF THE EMBODIMENTS
[0019] For the purposes of promoting an understanding of the
principles of the invention, reference will now be made to the
embodiments illustrated in the drawings and described in the
following written specification. It is understood that no
limitation to the scope of the invention is thereby intended. It is
further understood that the present invention includes any
alterations and modifications to the illustrated embodiments and
includes further applications of the principles of the invention as
would normally occur to one skilled in the art to which this
invention pertains.
Conductivity Cells
[0020] The present invention contemplates the integration of
conductivity cells, such as the cells 22, 24 depicted in FIG. 1,
within closed system 10 shown in FIG. 2 for removal of
cryoprotectant from a liquid, such as blood or a cell suspension.
As shown in FIG. 1, the conductivity cells 22, 24 include
flow-through conductivity electrodes 26 and 28 that are coupled to
tubing 30, 32. The tubing from part of the closed system 10, as
explained herein. The electrodes are separated by tubing 34 by a
predetermined distance to achieve a desired conductivity cell
constant K. In one specific embodiment, the electrodes are
stainless steel tubing, having a length of about 4.0 cm, an outer
diameter of about 0.48 cm. and an inner diameter of about 0.38 cm.
The tubing 34 is sized to separate the electrodes by about 9.0 cm
to achieve a cell constant of fifty. The high cell constant helps
minimize the effects of conductivity cell fouling. In the specific
example, the conductivity cells are configured to detect the
presence of DMSO in blood. Other cell constants may be preferable
for detecting other cryoprotectants in other liquids.
[0021] The electrodes are connected to a conductivity meter in a
conventional manner, such as with alligator clips. Alternatively,
the electrodes may incorporate contact points or wiring configured
to connect to a conductivity meter. One suitable conductivity meter
is the YSI.RTM. Model 32 meter. In addition, the electrodes 26, 28
may incorporate fluid fittings to facilitate connection to the
existing tubing 30, 32 of the closed system 10. For instance, the
ends of the electrodes 26, 28 may incorporate a barbed fitting.
Closed System for Removing Cryoprotectant from a
Cryoprotectant-Containing Liquid
[0022] The closed system 10 for removing cryoprotectant from a
liquid may be constructed as shown in FIG. 2 to incorporate the
dialyzer 12 disclosed in the '758 patent discussed above. Although
the specifics of the dialyzer 12 are found in the '758 patent, the
disclosure of which is incorporated herein by reference, certain
parts of the structure are shown in the present figure. In
particular, the dialyzer 12 includes a first inlet 13 and a first
outlet 14, as well as a second inlet 16 and a corresponding second
outlet 17. The first inlet 13 is connected to the source of the
cryoprotectant-containing liquid, while the second inlet 16 is
connected to a source of cryoprotectant-free liquid. The
corresponding outlets 14 and 17 discharge the liquids exiting the
dialyzer 12.
[0023] The closed system 10 shown in FIG. 2 provides a closed fluid
circuit commencing with fluid couplings C adapted to be coupled to
a liquid source, such as a blood bag B as shown in FIG. 3. One
coupling is connected to an outlet fluid line 50 through a valve
V2, which in turn is connected to the first inlet 13 of the
dialyzer 12 through a pump P1. The other coupling C is connected to
a return fluid line 52 which in turn is connected to the first
outlet 14 of the dialyzer 12 through a valve V3. A conductivity
cell 40 may be interposed between the return fluid line 52 and the
first outlet 13 of the dialyzer. As thus far described, the system
10 provides a closed fluid circuit that passes a
cryoprotectant-containing liquid through the dialyzer which returns
liquid to the source that has had the cryoprotectant level reduced
by the dialyzer 12.
[0024] The operation of the dialyzer 12 depends upon the passage of
a cryoprotectant-free liquid counter-flow to the flow of
cryoprotectant-containing liquid through the dialyzer. Thus, the
closed system 10 further includes a source S of a
cryoprotectant-free liquid, or dialysate, such as an isotonic salt
solution. This source S is connected to the second inlet 16 of the
dialyzer 12 by a dialysate line 54 through a valve V1. The
dialysate is discharged from the dialyzer 12 at second outlet 17
connected to dialysate discharge line 56. Since the dialysate
discharged from the dialyzer includes a quantity of cryoprotectant
removed from the other liquid passing through the device, the
discharged dialysate is sent to a waste container W. A pump P2 is
incorporated into the dialysate circuit to draw the dialysate
through the dialyzer 12. The pumps P1 and P2 are sized to achieve
an appropriate flow rate of the cryoprotectant-containing liquid
and the dialysate for optimum performance of the dialyzer. In a
specific example, the two pumps of the system 10 are adjustable
flow pumps capable of operating at a maximum flow rate of 150
ml/min.
[0025] A priming circuit is also provided to prime the dialyzer 12.
The circuit includes a source PS of priming solution with two
output lines 60 and 62. The output line 60 is connected through a
valve V5 to the dialysate line 54. The output line 62 is connected
through a valve V6 to the first inlet 13 of the dialyzer 12, or
more particularly to the outlet line 50 through which the
cryoprotectant-containing liquid is provided. The priming solution
flows through the dialyzer 12 prior to its operation on the
cryoprotectant-containing liquid.
[0026] In accordance with one aspect of the invention, a
differential conductivity device 20 is introduced into the
dialysate circuit. In particular the device 20 includes a
conductivity cell 22 connected across the outlet line 54 from the
dialysate source S, and a conductivity cell 24 connected across the
discharge line 56 prior to the pump P2 and waste container W. The
differential conductivity device 20 generates differential
conductivity readings for the dialysate entering and exiting the
dialyzer 12 during operation. A third conductivity cell 40 is
provided at the outlet 14 of the dialyzer 14 to measure the
conductivity of the cryoprotectant-containing liquid after it has
been treated in the dialyzer.
[0027] It is known that the readings produced by the conductivity
cells 22, 24 and 40 will vary with temperature. In order to obtain
accurate measurements the readings are temperature compensated.
Thus, a temperature probe is positioned close to each conductivity
cell. In particular, as shown in FIG. 2, a temperature sensor T1 is
adjacent cell 22, sensor T2 is adjacent cell 24 and temperature
sensor T3 adjacent cell 40. An additional temperature sensor T4 may
be provided at the blood inlet to the dialyzer 12.
Operation of the Closed System
[0028] Start-Up/Priming
[0029] At start-up, the system shown in FIG. 2 pumps a priming
solution into both parts of the dialyzer. The presence of the
priming solution in the various lines of the fluid circuits is
indicated by the notation "Priming". In one embodiment, the priming
solution is distilled water. Valve V6 is open to direct priming
solution from container PS into the inlet 13 of the dialyzer, while
valve V5 is open to direct priming solution into the dialysate
inlet 16. Valves V1 and V2 connected to the dialysate source S and
couplings C are both closed during priming of the dialyzer 12. The
priming solution is pumped through the dialyzer by pumps P1 and P2,
preferably at 150 ml/m. As shown in FIG. 2, the priming solution
fed into the dialysate inlet 16 first passes through conductivity
cell 22, while conductivity cell 24 receives the solution exiting
at the dialysate outlet 17. The priming solution exiting the
dialyzer at the first outlet 14 is directed by closed valve V3 and
open valve V4 to the waste container W for appropriate disposal.
Likewise, solution exiting through second outlet 17 is pumped into
the waste container.
[0030] As the priming solution flows through the system as shown in
FIG. 2, measurements at the conductivity cells 22, 24 and 40 are
temperature corrected. Correction factors are determined for cells
24 and 40 that adjust the measurements at these cells to be equal
to the measurement value of conductivity cell 24, since the same
liquid, the priming solution, is flowing through each cell In a
preferred embodiment, the temperature and conductivity values are
maintained within predetermined range and are permitted to vary
within predetermined narrow boundary values. Quick spikes in the
readings could be an indication of bubbles in the system. After
five minutes priming the dialyzer, the readings of the conductivity
cells should become stable.
[0031] Detecting Blood Cells
[0032] Once the dialyzer 12 has been primed, a blood bag B is
connected to the liquid couplings C, as shown in FIG. 3. In one
embodiment, the blood contains a cryoprotectant used in a cryogenic
process to store the blood sample. The cryoprotectant may be DMSO
and may be in solution in the blood in varying percentages. A
suitable dialysate to remove the DMSO is a phosphate buffered
saline (PBS). The dialyzer 12 is configured as disclosed in the
'758 patent to remove the DMSO, returning substantially pure blood
to the blood bag for subsequent use.
[0033] The state of the fluid circuit for blood detection within
the system 10 is illustrated in FIG. 3. When the blood bag is
attached, valve V2 is opened so blood solution is pumped into the
blood inlet 13 of the dialyzer by pump P1, as indicated by the
designation "Blood" in the system 10. The blood is preferably
pumped at 30 psi. When the blood first starts to flow into the
dialyzer, valve V3 is closed and valve V4 is opened to purge any
residual priming solution from the dialyzer blood circuit through
outlet 14 to the waste container W. The blood flowing through the
system has a higher concentration of DMSO than the priming solution
PBS. Thus, conductivity cell 40 will show the first indication of
the higher resistance of blood solution versus the priming solution
that had passed through the cell in the previous priming step shown
in FIG. 2.
[0034] The priming solution PBS is pumped through the dialysate
fluid circuit by the pump P2, since the valve V5 is open and the
valve V1 to the dialysate source S is closed. The resistance
measured by the conductivity cell 24 will also increase as the
blood starts through the system because blood will cross the
dialyzer into the priming solution present in the dialysate circuit
formed by fluid lines 54 and 56. Nominally, conductivity cell 40
will indicate a higher resistance than conductivity cell 24 because
the DMSO-containing blood transferred through the dialysis membrane
of the dialyzer will necessarily become diluted by the priming
solution already within the dialysate loop.
[0035] Blood Flow Back to Blood Bag
[0036] A change in measurement reading at conductivity cell 40
indicates when the blood has arrived at the cell, meaning that the
blood has passed through the dialyzer 12. At this point, the blood
flow is redirected back to the blood bag by closing valves V4 and
opening valve V3, as shown in FIG. 4. It should be noted that as in
the prior blood detection step, the priming solution continues to
be pumped through the dialysate circuit and into the waste
container at 150 psi. The priming solution is not adapted to
extract the DMSO from the blood.
[0037] As the blood continues to flow through the dialyzer, the
difference of the conductivity measurements between conductivity
cells 22 and 24 will approach a predetermined value that is near
zero. A near-zero differential conductivity means that the
dialysate circuit is fully primed and ready to receive the
dialysate from the source S.
[0038] When the differential conductivity between cells 22 and 24
reaches the predetermined value, valve V5 is closed to terminate
flow of priming solution through the system, as shown in FIG. 5.
(Valve V6 may also be closed). Valve V1 is then opened to direct
the dialysate PBS through the dialysate circuit of fluid lines 54
and 56, as indicated by the designation "Isotonic". When the
isotonic solution is flowed through the distillate side of the
dialyzer 12 the differential conductivity measurements will follow
the same pattern as for the priming solution. Thus, the difference
in measurements at conductivity cells 22 and 24 will show the
removal of the DMSO and the conductivity values registered by
conductivity cell 40 at the blood outlet 14 will reflect the
changes to DMSO concentration in the blood solution.
[0039] By continuing the flow of blood solution and isotonic
solution through the dialyzer as shown in the FIG. 5, the viable
cells in the blood solution can be concentrated. The integral of
the area under the temperature compensated conductivity curve for
conductivity cell 40 should indicate the concentration of DMSO-free
blood cells being returned to the blood bag. It is contemplated
that the measurement of the conductivity cell 40 will reach a
predetermined value indicative of removal of all or substantially
all of the DMSO from the blood solution.
[0040] Shut Down
[0041] The conductivity cells can be used to detect the process of
pushing viable cells from the dialyzer into the blood bag. In the
system 10 is configured to push substantially all of the available
viable cells into the blood bag using the priming solution. As
shown in FIG. 6, valve V2 is closed to terminate blood flow to
inlet 13, since the blood solution has been substantially purified.
Valve V6 is then opened so that priming solution will flow into the
inlet 13 behind the blood cells within the dialyzer 12. The
isotonic solution continues to flow through the distallate circuit
54, 56 and to the waste container W. This progress of the process
is indicated by conductivity changes in cells 24 and 40 to
determine when valve V3 should be closed. In other words, when the
conductivity measurements of conductivity cell 40 changes from the
conductivity value for purified blood to the conductivity value for
priming solution, it can be determined that all of the blood cells
have been pushed out of the dialyzer. A similar change will be
reflected at conductivity cell 24, although of a lesser magnitude,
as blood cross-over into the dialysate circuit ceases.
[0042] Flow Reversal
[0043] In a final step, the flow of pump P1 is reversed to pushing
any blood cells remaining in the outlet line 50 back into the blood
bag B. Thus, as shown in FIG. 7, valve V2 is opened, pump P1 is
reversed, and valve V4 is opened to permit flow through the pump
behind the blood cells. Flow of the isotonic solution through the
dialyzer is ceased by stopping pump P2, while valves V5 and V6 are
closed to terminate any priming solution flow. Thus, there will be
no indication of activity at the conductivity cells 22 and 24.
However, conductivity cell 40 will measure the conductivity changes
from isotonic solution to priming solution, which may be used to
indicate a stopping point for the process. Alternatively, the
duration of the process may be limited to a predetermined time
believed to be sufficient to return all viable blood cells to the
blood container. This can be achieved in one embodiment by counting
steps of the stepper motor driving pump P1, or by a direct time
measurement to indicate when to de-energize the pump.
[0044] In certain cases, some of the blood returning to the blood
bag through fluid line 52 can be immediately drawn back through the
system through outlet line 50. This phenomenon can lead to a false
indication of a reduction in DMSO in the blood. This occurrence can
be prevented by segmenting the blood bag B that will prevent the
returning "clean" blood from displacing the untreated blood
remaining in the bag. In one approach, a baffle is created between
the ports on the blood bag to prevent any cross-contamination.
Other approaches may be implemented to ensure that all of the
untreated blood in the blood bag B flows through the closed system
10 before the conductivity cells indicate a DMSO level indicative
that the DMSO has been substantially removed from the blood.
[0045] It is contemplated that the sequence of opening and closing
the valves V1-V6 and activating/de-activating pumps P1 and P2 can
be controlled by a master controller. The controller receives
signals from the conductivity cells 22, 24 and 40, as well as from
the temperature sensors T1-T3, and generates control signals for
the opening and closing the valves and for energizing,
de-energizing and controlling flow rate and direction of the pumps
P1 and P2. The controller may be analog with appropriate circuitry
to evaluate measurement differences between cells and between cell
measurements and threshold values.
[0046] Preferably, the components are digital and the controller is
a programmable microcontroller. The digital controller can adjust
the conductivity measurements generated by each conductivity cell
in relation to the temperature measured by the adjacent temperature
sensor. Adjusted values for the conductivity cells 22 and 24 can be
used to generate the differential conductivity values used to
determine when the cryoprotectant has been substantially removed
from the blood. The adjusted values of all the cells can also be
compared as described above to determine when the priming step has
completed or when substantially all of the blood cells have been
returned to the blood bag B.
[0047] The controller can also be configured to compare the
differential and actual conductivity values to the various
thresholds used to determine when one step is complete and another
is to begin. The controller may permit user input to change the
threshold values based on a particular cryoprotectant,
cryoprotectant-containing liquid, dialysate or priming solution.
Alternatively, or additionally, the controller may incorporate a
data base of stored values that can be selected by identifying the
particular combination of liquids. In addition to storing threshold
values, the controller may also store the desired pump flow rates
for the various steps of operation of the system 10.
[0048] It is further contemplated that the controller may be
configured to monitor the rate of change in conductivity or
differential conductivity at the differential conductivity device
20 as an indication of the rate or removal of cryoprotectant from
the blood. In some circumstances, removal that occurs to rapidly
can damage the blood cells. Thus, when the rate of change of
conductivity or differential conductivity exceeds a predetermined
threshold value the controller can alter the flow through the
system 10, such as by reducing the flow rate of one or both of the
pumps P1 and P2, to thereby protect the blood cells from rapid
osmolality change.
[0049] Conductivity tests using the conductivity cell disclosed
herein have been conducted to establish baseline values for certain
substances and to evaluate the change in these values with
temperature. For instance, tests for distilled water show a
conductivity of 1.10 E-07 mohs at 10.degree. C. and an essentially
linear 2.43% change in conductivity per each degree of temperature
change. Similar tests on a 10% DMSO solution show initial
conductivity of 5.34 E-02 mohs and a rate of change of 2.10%. The
conductivity at 10.degree. C. increases to 6.48 E-02 mohs for a
2.5% DMSO solution while the rate of change with temperature
decreases to 1.97%. Similar tests for a 4.5% PBS solution show a
conductivity at 10.degree. C. of 3.93 E-04 mohs and a rate of
change of 1.76%, with the conductivity decreasing to 7.20 E-05 mohs
and rate of change increasing to 1.84% for a 0.9% PBS solution. The
conductivity values for sucrose are 3.50 E-07 mohs and 2.40%.
[0050] These test results reveal a difference in magnitude of
conductivity values among the various substances flowing through
the closed system 10 and the differential conductivity device 20.
Thus, the differential conductivity readings used to determine when
the blood is substantially free of cryoprotectant will be very
pronounced initially since the lower conductivity pure dialysate
PBS will be flowing through the cell 22 and the much higher
conductivity PBS-DMSO solution exiting the dialyzer 12 will be
flowing through the second cell 24. The amount of DMSO purged from
the blood flowing through the dialyzer 12 decreases with each pass
of the blood through the system so that the conductivity of the
liquid flowing through the second cell 24 will gradually decrease
to match the conductivity of the pure dialysate PBS at cell 22.
[0051] The closed system 10, the differential conductivity device
20 and the conductivity cell 40, as well as the protocol disclosed
herein, can be used very effectively to remove cryoprotectant from
a quantity of blood that has been previously frozen and then
thawed. The same system and protocol can be used to remove other
cryoprotectants from blood, such as glycerol, provided the
conductivity values are different enough from the baseline liquids
and/or that the conductivity cells are sensitive enough to measure
more subtle differences in conductivity. Moreover, the present
system 10 and the protocol described above can be used to monitor
the removal of other substances from a particular liquid, again
provided the conductivity values and change in conductivity are
readily detectable by the conductivity cells.
[0052] While the invention has been illustrated and described in
detail in the drawings and foregoing description, the same should
be considered as illustrative and not restrictive in character. It
is understood that only the preferred embodiments have been
presented and that all changes, modifications and further
applications that come within the spirit of the invention are
desired to be protected.
* * * * *