NZ603760B - Leukocyte purification - Google Patents
Leukocyte purification Download PDFInfo
- Publication number
- NZ603760B NZ603760B NZ603760A NZ60376012A NZ603760B NZ 603760 B NZ603760 B NZ 603760B NZ 603760 A NZ603760 A NZ 603760A NZ 60376012 A NZ60376012 A NZ 60376012A NZ 603760 B NZ603760 B NZ 603760B
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- Prior art keywords
- leukocytes
- fluid
- leukocyte depletion
- passing
- leukocyte
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0081—Purging biological preparations of unwanted cells
- C12N5/0087—Purging against subsets of blood cells, e.g. purging alloreactive T cells
Abstract
Patent 603760 Disclosed is a method for purifying leukocytes, the method comprising (a) passing a biological fluid comprising leukocytes, or a cryoprotectant fluid comprising leukocytes, through a porous leukocyte depletion medium having an upstream surface and a downstream surface, the fluid passing from the upstream surface through the downstream surface, wherein leukocytes are retained by the leukocyte depletion medium; (b) passing a wash fluid through the leukocyte depletion medium from the downstream surface through the upstream surface; and, (c) passing the wash fluid through the leukocyte depletion medium from the upstream surface through the downstream surface via gravity. passing from the upstream surface through the downstream surface, wherein leukocytes are retained by the leukocyte depletion medium; (b) passing a wash fluid through the leukocyte depletion medium from the downstream surface through the upstream surface; and, (c) passing the wash fluid through the leukocyte depletion medium from the upstream surface through the downstream surface via gravity.
Description
COMPLETE SPECIFICATION
LEUKOCYTE PURIFICATION
LEUKOCYTE PURIFICATION
BACKGROUND OF THE INVENTION
Leukocytes (also referred to as white blood cells) are useful in a variety of
applications, including cell therapy and diagnostics. Leukocytes can be obtained from, for
example, cord blood or blood obtained from a donor.
Conventionally, cord blood concentrates and stem cell concentrates, which
include leukocytes, are stored under liquid nitrogen conditions, wherein the concentrates
include the cryoprotectant dimethyl sulfoxide (DMSO) to protect the leukocytes from
damage during storage. The concentrates including DMSO can be administered to a patient,
but since DMSO can be considered an impurity, some protocols include centrifuging and
washing the concentrates to reduce the presence of DMSO in the concentrate product before
administration to a patient. Such protocols are labor intensive and time consuming.
Some methods for obtaining leukocytes include passing blood or blood products
through a porous leukocyte depletion medium and either eluting the leukocytes from the
medium by passing an elution fluid from the downstream surface of the medium through the
upstream surface; or lysing the leukocytes on the medium after forcing saline through the
medium from the upstream surface through the downstream surface or forcing air through the
medium from the upstream surface through the downstream surface. However, these
methods have resulted in an undesirable level of impurities, such as the presence of
erythrocytes (also known as red blood cells) and/or a lower than desired yield of leukocytes.
It is an object of the present invention to provide for ameliorating at least some of
the disadvantages of the prior art and/or to provide the public with a useful choice. These and
other advantages of the present invention will be apparent from the description as set forth
below.
BRIEF SUMMARY OF THE INVENTION
In a first embodiment, the invention provides a method for purifying leukocytes,
the method comprising (a) passing a biological fluid comprising leukocytes through a porous
leukocyte depletion medium having an upstream surface and a downstream surface, the fluid
passing from the upstream surface through the downstream surface, wherein leukocytes are
retained by the leukocyte depletion medium; (b) passing a wash fluid through the leukocyte
depletion medium from the downstream surface through the upstream surface; and, (c)
passing the wash fluid through the leukocyte depletion medium from the upstream surface
through the downstream surface via gravity.
Another embodiment describes a method for purifying leukocytes, the method
comprising (a) passing a cryoprotectant fluid comprising leukocytes through a porous
leukocyte depletion medium having an upstream surface and a downstream surface, the fluid
passing from the upstream surface through the downstream surface, wherein leukocytes are
retained by the leukocyte depletion medium; (b) passing a wash fluid through the leukocyte
depletion medium from the downstream surface through the upstream surface; and, (c)
passing the wash fluid through the leukocyte depletion medium from the upstream surface
through the downstream surface via gravity.
In some embodiments of the methods, (b) and (c) are repeated at least once with
additional wash fluid.
Some embodiments of the methods further comprise eluting leukocytes from the
leukocyte depletion medium by passing an elution fluid through the leukocyte depletion
medium from the downstream surface through the upstream surface, and recovering the
eluted leukocytes, or lysing the leukocytes while retained by the leukocyte depletion medium.
In a second embodiment, the invention provides leukocyte cells prepared by the
method of the invention.
In a third embodiment, the invention provides a use of the leukocyte of the
invention in the manufacture of a medicament
In a fourth embodiment, the invention provides a use of the leukocytes of the
invention, in the manufacture of a medicament for treating a disease or disorder responsive
thereto in a subject in need thereof.
In a fifth embodiment, the invention provides nucleic acids produced by the
method of the invention.
In a sixth embodiment, the invention provides lysed leukocytes produced by the
method of the invention.
If desired, embodiments of the method can be carried out using multiwell device,
or individual filter devices.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
Figure 1 describes, and illustrates diagrammatically, an embodiment of a method
according to the present invention.
Figure 2 shows an illustrative system suitable for carrying out an embodiment of a
method according to the invention.
Figure 3 shows another illustrative system suitable for carrying out an
embodiment of a method according to the invention, wherein the method is carried out while
maintaining a closed system.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with an embodiment of the present invention, a method for
purifying leukocytes is provided, the method comprising (a) passing a biological fluid
comprising leukocytes through a porous leukocyte depletion medium having an upstream
surface and a downstream surface, the fluid passing from the upstream surface through the
downstream surface, wherein leukocytes are retained by the leukocyte depletion medium; (b)
passing a wash fluid through the leukocyte depletion medium from the downstream surface
through the upstream surface; and, (c) passing the wash fluid through the leukocyte depletion
medium from the upstream surface through the downstream surface via gravity.
Another embodiment of a method for purifying leukocytes comprises (a) passing
a fluid comprising leukocytes and a cryoprotectant (such as DMSO) through a porous
leukocyte depletion medium having an upstream surface and a downstream surface, the fluid
passing from the upstream surface through the downstream surface, wherein leukocytes are
retained by the leukocyte depletion medium; (b) passing a wash fluid through the leukocyte
depletion medium from the downstream surface through the upstream surface; and, (c)
passing the wash fluid through the leukocyte depletion medium from the upstream surface
through the downstream surface via gravity.
In some embodiments of the methods, (b) and (c) are repeated at least once with
additional wash fluid.
In some embodiments, the biological fluid or cryoprotectant fluid is passed
through the porous leukocyte depletion medium via gravity or vacuum. Passing the wash
fluid through the leukocyte depletion medium from the downstream surface through the
upstream surface comprises creating a negative pressure (vacuum) upstream of the upstream
surface, or comprises creating a positive pressure downstream of the downstream surface.
Advantageously, leukocytes can be obtained while reducing the presence of red
blood cells and/or platelets. Minimizing the presence of red blood cells and platelets is
particularly advantageous for cell therapy applications. Alternatively, or additionally,
minimizing the presence of DMSO is particularly advantageous for cell therapy applications,
especially for those involving neonatals, infants, and children.
Some embodiments of the methods further comprise eluting leukocytes from the
leukocyte depletion medium by passing an elution fluid through the leukocyte depletion
medium from the downstream surface through the upstream surface, and recovering the
eluted leukocytes, or lysing the leukocytes while retained by the leukocyte depletion medium.
In one embodiment including lysing the leukocytes, the method further comprises analyzing
the content of the lysed leukocytes, e.g., comprising analyzing nucleic acids from the lysed
leukocytes.
In one preferred embodiment of the method, the eluted leukocytes are
administered to a subject, such as a patient.
If desired, embodiments of the method can be carried out using multiwell device,
or individual filter devices. For example, using a multiwell device having a plurality of wells
having leukocyte depletion media therein, separate portions of biological fluid and wash fluid
are passed through the leukocyte depletion media in separate wells.
The invention can be carried out using biological fluid from a variety of sources,
particularly mammals (the cells in the cryoprotectant fluid can also be from a variety of
sources). It is preferred that the mammals are from the order Carnivora, including Felines
(cats) and Canines (dogs), the order Artiodactyla, including Bovines (cows) and Swines
(pigs) or of the order Perssodactyla, including Equines (horses). Typically, the mammals are
of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans
and apes). An especially preferred mammal is the human.
In accordance with embodiments of the invention, any suitable volume of
biological fluid or cryoprotectant fluid can be processed, and a variety of leukocyte depletion
media and leukocyte depletion filter configurations are suitable, e.g., media or filters having
diameters in the range from, for example, about 0.3 inches (about 7 mm) or less, to about 5
inches (about 12 cm), or more.
The cord blood concentrates and stem cell concentrates used in accordance with
the invention can be produced (e.g., to obtain an enriched white cell fraction, combined with
a cryoprotectant agent, frozen, and thawed) by a variety of techniques known in the art before
the leukocytes are purified (and, preferably, eluted) in accordance with embodiments of the
invention.
Each of the components of the invention will now be described in more detail
below, wherein like components have like reference numbers.
A variety of porous leukocyte depletion media are suitable for use in the
invention. Suitable leukocyte depletion media include commercially available media, for
example, media available from Pall Corporation (Port Washington, NY).
A variety of wash fluids are suitable for use in the invention. Typically, the wash
fluid is physiologically compatible with the desired cells, and does not substantially effect the
cells. Illustrative fluids include, for example, saline, for example, phosphate buffered saline
(PBS).
Washing can be accomplished at any suitable fluid flow rate. Typically, passing
the wash fluid through the leukocyte depletion medium from the downstream surface through
the upstream surface comprises creating a negative pressure upstream of the upstream
surface, e.g., by withdrawing the plunger in a syringe barrel, wherein the syringe barrel is
connected to the inlet of the filter device, or creating a vacuum in the well(s) of a multiple
well device. Preferably, passing the wash fluid through the leukocyte depletion medium from
the upstream surface through the downstream surface is accomplished via gravity.
In those embodiments including elution, the desired retained cells, i.e., leukocytes
(and in some embodiments, stem cells) retained (e.g., captured and/or adsorbed) by the filter
are released by backflushing from the porous leukocyte depletion medium, i.e., by passing
the elution fluid through the porous medium in a direction from the downstream side towards
the upstream side, and through the inlet port, such that the elution fluid containing the cells is
passed from the inlet port into a cell collection container communicating with the inlet port.
The backflushing can be accomplished at any suitable fluid flow rate, e.g., about
0.1-15 L/min/m , although flow rates significantly more or less than this range can be used.
For example, backflushing can be accomplished at a fluid flow rate of about 0.5-10
L/min/m , such as about 1-8 L/min/m ; more preferably the flow rate is about 1.5-7
2 2 2
L/min/m , such as about 2-6 L/min/m or even about 2.5-5 L/min/m (e.g., about 3-4 L
ml/min/m ). The most preferable flow rate may depend upon the viscosity and/or the
temperature of the elution fluid, and the nature of the filter medium. Thus, in some
applications, such as when more gentle treatment is desired, backflushing can be
accomplished at a flow rate about 1-100 ml/min/m , (e.g., about 15-85 ml/min/m ); more
preferably the flow rate is about 30-70 ml/min/m or even about 40-60 ml/min/m (e.g., about
50 ml/min/m ).
A variety of elution fluids are suitable for use in the invention. Typically, the
fluid is physiologically compatible with the desired cells, and does not substantially effect the
cells. Illustrative fluids include, for example, saline, as well as those fluids, including more
viscous fluids, disclosed in U.S. Patents 6,544,751 and 7,291,450.
In those embodiments wherein leukocytes are purified in accordance with the
invention, and are subsequently lysed in or on the leukocyte depletion medium (rather than
eluted from the medium), a variety of reagents and protocols are suitable for treating and
lysing the leukocytes, and purifying and/or isolating the nucleic acids (preferably, RNA, but
embodiments of the invention include purifying and/or isolating DNA) released from the
leukocytes and are known in the art. If desired, the leukocytes can be treated to stabilize the
cells before lysing. The stabilized cells can be maintained in or on the leukocyte depletion
medium for a suitable period before lysis, or the cells can be lysed shortly or immediately
after stabilization. Preferably, the leukocytes are disputed using a solution such that RNA is
rapidly released while inactivating nucleases. Illustrative reagents and protocols, including
purifying RNA, isolating total RNA, and depleting globin RNA from purified RNA, are
disclosed in, for example, “LeukoLOCK™ Total RNA Isolation System, Globin
mRNA-Depleted Total RNA from Whole Blood Samples,” Instruction Manual, Catalog #AM
1923, AM1933, AM1934, (Manual 1923M Revision B) Ambion Inc., Austin, TX (2007).
A variety of containers and devices comprising containers are suitable for use in
the invention, wherein the containers can be arranged upstream and/or downstream of the
leukocyte depletion media. Suitable containers are known in the art. Typically, one or more
of the containers are physiologically compatible with the biological fluid, the desired cells,
the wash fluids, and/or the elution fluids, and do not substantially effect the cells. Since a
plurality of containers can be used in embodiments of the invention, it is not a requirement
that a single container be physiologically compatible with the biological fluid, the desired
cells, the wash fluids, and the elution fluids. For example, one container can be used for
passing biological fluid or cryoprotectant fluid through the leukocyte depletion medium (and
thus need not be physiologically compatible with wash fluid and/or elution fluid), another can
be used for receiving wash fluid through the leukocyte depletion medium and passing wash
fluid through the leukocyte depletion medium, and another can be used for receiving eluted
cells and elution fluid.
In some embodiments, the container(s) used for passing biological fluid or
cryoprotectant fluid through the leukocyte depletion medium, and receiving wash fluid
through the leukocyte depletion medium and passing wash fluid through the leukocyte
depletion medium, is/are substantially non-flexible, e.g., non-flexible containers such as
syringe barrels. However, embodiments of the invention are not so limited. For example, the
container(s) used for passing biological fluid or cryoprotectant fluid through the leukocyte
depletion medium, and receiving wash fluid through the leukocyte depletion medium and
passing wash fluid through the leukocyte depletion medium can comprise flexible containers
such as blood bags (e.g., plasticized blood bags), having flexible side walls, typically,
wherein the container(s) has/have at least two ports, e.g., an inlet port and an outlet port. The
flexible side walls can be compressed or a vacuum can be created such that air and/or liquid
passes from the container and/or the flexible side walls expand when air and/or liquid enters
the bag and the bag is pressurized, and the compliant container directs the air and/or liquid
out when the outlet port is opened.
Embodiments of the invention are suitable for use in open systems, and
embodiments of the invention can be carried out while maintaining closed systems.
The following definitions are used in accordance with the invention.
Biological Fluid. A biological fluid includes any treated or untreated fluid
associated with living organisms, particularly blood, including whole blood, warm or cold
blood, cord blood, and stored or fresh blood; treated blood, such as blood diluted with at least
one physiological solution, including but not limited to saline, nutrient, and/or anticoagulant
solutions; blood components, such as platelet concentrate (PC), platelet-rich plasma (PRP),
platelet-poor plasma (PPP), platelet-free plasma, plasma, fresh frozen plasma (FFP),
components obtained from plasma, packed red cells (PRC), transition zone material or buffy
coat (BC); blood products derived from blood or a blood component or derived from bone
marrow; stem cells; cord blood; red cells separated from plasma and resuspended in a
physiological solution or a cryoprotective fluid; and platelets separated from plasma and
resuspended in a physiological solution or a cryoprotective fluid. A biological fluid also
includes a physiological solution comprising a bone marrow aspirate. The biological fluid
may have been treated to remove some of the leukocytes before being processed according to
the invention. As used herein, blood product or biological fluid refers to the components
described above, and to similar blood products or biological fluids obtained by other means
and with similar properties.
A “cryoprotectant fluid” can include, for example, a cryoprotectant agent and
mixtures thereof, such as, but not limited to dimethyl sulfoxide (DMSO), glycerol,
polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, I-
erythritol, D-ribitol, D-mannitol, D-sorbitol, I-inositol, D-lactose, choline chloride, amino
acids, methanol, acetamide, glycerol monoacetate, and inorganic salts. Exemplary treatment
solutions include those disclosed in International Publication No. WO 96/17514. Typically,
embodiments, DMSO is used, which is nontoxic to cells in low concentration. It is believed
that, being a small molecule, DMSO freely permeates the cell and protects intracellular
organelles by combining with water to modify its freezability and prevent damage from ice
formation. The addition of plasma (e.g., to a concentration of about 20-25%) can augment
the protective effect of DMSO.
A "unit" is the quantity of biological fluid from a donor or derived from one unit
of whole blood. It may also refer to the quantity drawn during a single donation. Typically,
the volume of a unit varies, the amount differing from patient to patient and from donation to
donation. Multiple units of some blood components, particularly platelets and buffy coat,
may be pooled or combined, typically by combining four or more units.
As used herein, the term “closed” refers to a system that allows the collection and
processing (and, if desired, the manipulation, e.g., separation of portions, separation into
components, filtration, storage, and preservation) of cryoprotectant fluid or biological fluid,
e.g., donor blood, blood samples, and/or blood components, without the need to compromise
the sterile integrity of the system. A closed system can be as originally made, or result from
the connection of system components using what are known as “sterile docking” devices.
Illustrative sterile docking devices are disclosed in, for example, U.S. Patents 4,507,119,
4,737,214, and 4,913,756.
A variety of materials can be used, including synthetic polymeric materials, to
produce the porous leukocyte depletion media (preferably, fibrous porous leukocyte depletion
media) of the filter elements according to the invention. Suitable synthetic polymeric
materials include, for example, polybutylene terephthalate (PBT), polyethylene, polyethylene
terephthalate (PET), polypropylene, polymethylpentene, polyvinylidene fluoride,
polysulfone, polyethersulfone, nylon 6, nylon 66, nylon 6T, nylon 612, nylon 11, and nylon 6
copolymers, wherein polyesters, e.g., PBT and PET, are more preferred. Typically, the
fibrous porous media are prepared from melt-blown fibers. For example, U.S. Patents
4,880,548; 4,925,572, 5,152,905, and 6,074,869, disclose porous leukocyte depletion filters
and filter elements prepared from melt-blown fibers.
A leukocyte depletion filter element can have any suitable pore structure, e.g., a
pore size (for example, as evidenced by bubble point, or by K as described in, for example,
U.S. Patent 4,340,479, or evidenced by capillary condensation flow porometry), a pore rating,
a pore diameter (e.g., when characterized using the modified OSU F2 test as described in, for
example, U.S. Patent 4,925,572), or removal rating that reduces or allows the passage
therethrough of one or more materials of interest as the fluid is passed through the element.
While it is believed leukocytes are primarily removed by adsorption, they can also be
removed by filtration. The pore structure can be selected to remove at least some level of
leukocytes, while allowing the passing therethrough of other components, e.g., plasma,
platelets, and red blood cells. The pore structure used depends on the composition of the
fluid to be treated, and the desired effluent level of the treated fluid.
The filter element can have any desired critical wetting surface tension (CWST, as
defined in, for example, U.S. Patent 4,925,572). The CWST can be selected as is known in
the art, e.g., as additionally disclosed in, for example, U.S. Patents 5,152,905, 5,443,743,
,472,621, and 6,074,869. Typically, the filter element has a CWST of greater than about 53
dynes/cm (about 53 x 10 N/cm), more typically greater than about 58 dynes/cm (about 58 x
-5 -5
N/cm), and can have a CWST of about 66 dynes/cm (about 66 x 10 N/cm) or more. In
some embodiments, the element has a CWST of 75 dynes/cm (about 75 x 10 N/cm) or
more, e.g., in the range of about 80 to about 100 dynes/cm (about 80 to about 100 x 10
N/cm).
The surface characteristics of the element can be modified (e.g., to affect the
CWST, to include a surface charge, e.g., a positive or negative charge, and/or to alter the
polarity or hydrophilicity of the surface) by wet or dry oxidation, by coating or depositing a
polymer on the surface, or by a grafting reaction. Modifications include, e.g., irradiation, a
polar or charged monomer, coating and/or curing the surface with a charged polymer, and
carrying out chemical modification to attach functional groups on the surface. Grafting
reactions may be activated by exposure to an energy source such as gas plasma, vapor
plasma, corona discharge, heat, a Van der Graff generator, ultraviolet light, electron beam, or
to various other forms of radiation, or by surface etching or deposition using a plasma
treatment.
The filter and/or filter device can include additional elements, layers, or
components, that can have different structures and/or functions, e.g., at least one of
prefiltration, support, drainage, spacing and cushioning.
The filter element, in some embodiments a filter comprising a plurality of filter
elements, can be disposed in a housing comprising at least one inlet and at least one outlet
and defining at least one fluid flow path between the inlet and the outlet, wherein the filter
element is across the fluid flow path, to provide a filter device. Typically, the filter device is
sterilizable. Any housing of suitable shape and providing at least one inlet and at least one
outlet may be employed.
Alternatively, filter elements or filters comprising one or more filter elements can
be disposed in multiple well devices or filter plates, wherein filter elements or filters
comprising a plurality of filter devices are disposed in the wells. Suitable devices and filter
plates are known in the art and are commercially available, e.g., from Pall Corporation (Port
Washington, NY) under the tradenames ACROWELL and ACROPREP and/or described
in, for example, International Publication No. .
The housing can be fabricated from any suitable rigid impervious material,
including any impervious thermoplastic material, which is compatible with the biological
fluid being processed. Typically, the housing is fabricated from a polymer. In some
embodiments, the polymer a transparent or translucent polymer, such as an acrylic,
polypropylene, polystyrene, or a polycarbonated resin. Such a housing is easily and
economically fabricated, and allows observation of the passage of fluid through the housing.
If desired, the housing can be sealed as is known in the art, utilizing, for example,
an adhesive, a solvent, laser welding, radio frequency sealing, ultrasonic sealing and/or heat
sealing. Additionally, or alternatively, the housing can be sealed via injection molding.
The following examples further illustrate the invention but, of course, should not
be construed as in any way limiting its scope.
EXAMPLE 1
This example demonstrates leukocytes can be purified and recovered with a good
yield and while minimizing red blood cell contamination according to an embodiment of the
invention.
The filter devices are 25 mm ACRODISC® syringe filters with Leukosorb
leukocyte depletion media (Pall Corporation, Port Washington, NY). The inlet ports of the
devices are connected to empty 5 mL syringe barrels, the outlet ports are connected to tubing
leading to a waste container, and 9 mL samples of freshly collected blood from donors are
treated with EDTA or CP2D anticoagulant, drain, via gravity, through the filters. The filtered
blood is discarded.
New 5 mL syringe barrels (including plungers) are connected to the inlet ports of
the devices, and the tubing connected to the outlet ports of the devices are placed in
containers containing about 8 μL of phosphate buffered saline (PBS) (“the waste
containers”). As shown in Figure 1, an empty syringe barrel, connected to the inlet of a
syringe filter including a leukocyte depletion medium, is arranged such that the outlet of the
syringe filter is in fluid communication with a receiving container. Leukocyte-containing
biological fluid is placed in the barrel, and drains through the filter via gravity, wherein
leukocytes are captured by the leukocyte depletion medium.
The syringe barrel is disconnected from the syringe filter inlet, a syringe plunger
is inserted in the barrel, and the syringe is reconnected to the inlet. The outlet of the syringe
filter is placed in fluid communication with a wash container containing was fluid. The
plunger is retracted, withdrawing wash fluid from the wash container, through the filter, and
into the syringe barrel.
Retraction of the syringe plunger continues until it is pulled out of the barrel. Was
solution drains through the filter back into the wash container via gravity. Leukocytes are re-
captured by the leukocyte depletion medium, and undesired material (e.g., red blood cells)
passes into the wash container.
Washing can be repeated with fresh wash fluid.
Thus, as shown in Figure 1, the syringe plungers are withdrawn to pull PBS from
the waste containers, through the downstream surfaces of the leukocyte depletion media and
the upstream surfaces, and into the syringe barrels. After the PBS is pulled into the syringe
barrels, the syringe plungers are pulled out of the barrels, while the device outlets remain in
contact with the PBS in the waste containers. The PBS in each syringe barrel then drains, via
gravity through the upstream surface and the downstream surface, and into the waste
container. The wash is repeated, as another volume of PBS is pulled through the media and
allowed to drain as described above.
5 mL of elution solution comprising 6% dextran 70 in PBS as generally described
in U.S. Patent 6,544,751 is passed from the outlet of each device through the inlet, and the
eluted leukocytes are recovered.
Influent, effluent, and eluted leukocyte and red blood cell counts are taken. The
results are as follows:
Sample Anti- Trapped Recovered Residual RBCs
coagulant WBCs, WBCs, in the WBCs
% to Control % to Control eluate, % to Control
1 EDTA 89 61 0.1
2 EDTA 96 77 0.03
3 CP2D 88 62 0.01
4 CP2D 96 84 0.02
EDTA 86 71 0.08
6 CP2D 96 70 0.05
7 EDTA 90 80 0.1
8 CP2D 89 81 0.03
9 EDTA 87 63 0.1
Table 1
In each sample, compared to the controls, over 85% of the white blood cells are
trapped and over 60% of the white blood cells compared to the control are recovered, and,
compared to the controls, 1% or less red blood cells are present in the eluted while blood
cells.
EXAMPLE 2
This example demonstrates the reduction in red blood cell contamination while
purifying and recovering leukocytes according to an embodiment of the invention.
The system 100 used for carrying out this Example is shown in Figure 2, showing
a filter device 1, having an inlet port 2 and an outlet port 3, source bag (blood bag) 4, waste
bag 6, leukocyte sampling bag 9, syringe 7, injection port 8, and clamps 10-13.
The filter devices are PURECELL® PXLA High Efficiency Leukocyte Reduction
Filters for Apheresis Platelet Transfusion with leukocyte depletion media (Pall Corporation,
Port Washington, NY). The inlet port 2 of the filter device 1 is connected through tubing and
a “Y” connector to the blood bag 4 and to the leukocyte sampling bag 9, the device outlet
port 3 is connected to tubing leading through another “Y“ connector to the waste bag 6 and to
the injection port 8, which is connected to syringe 7.
The syringe is filled with elution solution and attached to port 8 while clamp 12 is
closed.
In order to control the flow, clamps are placed on the tubing between the inlet port
and the blood bag and the sampling bag (clamps 10 and 11), and between the outlet port and
the waste bag and the injection port (clamps 12 and 13). All the clamps are initially closed.
The blood bag contains 50 mL of blood collected from donors and mixed with
anticoagulant according to a standard blood bank procedure. The system is arranged
vertically, and clamps 10 and 13 are opened, blood is filtered via gravity, passing into waste
container 6, and leucocytes are captured on the leukocyte depletion medium in the filter
device 1.
Clamp 13 is closed, clamp 12 is opened and 20 mL of PBS wash solution is
injected via syringe 7 through injection port 8, such that the wash solution passes through the
downstream surface of the leukodepletion medium and through upstream surface and into the
blood bag 4. Clamps 10 and 13 are opened again and wash solution is allowed to drain via
gravity through the filter device into the waste bag 6.
The wash procedure is repeated once more with fresh PBS.
The syringe 7 is filled with elution solution (6% dextran 40 in PBS) and connected
to the injection port 8.
Clamps 10 and 13 are closed, and clamps 11 and 12 are opened, and 24 uL of the
elution solution is injected from the syringe 7 into the injection port 8 such that leukocytes
are eluted and pass, with the elution solution, into the sampling bag 9.
For one experiment, the wash fluid is not drained into a waste bag, for the other
experiment, the wash fluid is drained into waste bag 6. Additionally, leukocytes are
recovered without the use of a wash, and the results for the various filtrations are compared.
The results are as follows (percent recovery into elution buffer):
wash WBC lymphocytes monocytes granulocytes RBC platelets
none 59 75 77 50 15 25
2x 65 77 86 56 3.6 18
2x drain 62 70 80 56 3.4 19
into waste
The results show using the wash reduced red blood cell contamination by over
66% as compared to no wash.
EXAMPLE 3
This example demonstrates that leukocytes can be purified and lysed and the
leukocyte nucleic acids can be analyzed according to an embodiment of the invention.
The filter devices are 25 mm Acrodisc® syringe filters with Leukosorb leukocyte
depletion media (Pall Corporation, Port Washington, NY).
9 mL samples of freshly collected blood from donors are treated with EDTA
anticoagulant, and processed as follows. Influent and effluent leukocyte counts are taken.
For the first experiment, the inlet ports of the devices are connected to empty 5
mL syringe barrels, the outlet ports are connected to tubing leading to a waste container, and
9 mL samples of freshly collected blood from donors were treated with EDTA anticoagulant,
drain, via gravity, through the filters. The filtered blood is discarded.
New 5 mL syringe barrels (including plungers) are connected to the inlet ports of
the devices, and the tubing connected to the outlet ports of the devices are placed in waste
containers containing about 8 μL of phosphate buffered saline (PBS). The syringe plungers
are withdrawn to pull PBS from the containers, through the downstream surfaces of the
leukocyte depletion media and the upstream surfaces, and into the syringe barrel. After the
PBS is pulled into the syringe barrels, the syringe plungers are pulled out of the barrels, while
the device outlets remain in contact with the PBS in the waste containers. The PBS in each
syringe barrel then drains, via gravity through the upstream surface and the downstream
surface, and into a waste container. The wash is repeated, as another volume of PBS is pulled
through the media and allowed to drain as described above.
2.5 mL of pH-adjusted Lysis/Binding Solution is passed through the medium
using a syringe, and collected in a lysate collection tube. Lysate is added with 2.5 mL of
Nuclease-free Water, and Proteinase K. Total RNA is isolated as described in
“LeukoLOCK™ Total RNA Isolation System, Globin mRNA-Depleted Total RNA from
Whole Blood Samples,” Instruction Manual, Catalog #AM 1923, AM1933, AM1934,
(Manual 1923M Revision B) Ambion Inc., Austin, TX (2007) (the “LeukoLOCK™ Total
RNA Isolation System Manual”).
In the second experiment, the blood is passed through the filter following the
procedure as described in the LeukoLOCK™ Total RNA Isolation System Manual, and the
leukocytes are isolated, stabilized, lysed, and the total RNA is isolated as described in the
LeukoLOCK™ Total RNA Isolation System Manual.
The efficiency of leukocyte capture, the RNA yield, and the optical density ratio
of absorbance at 260 and 280 nm (A ) is determined in each experiment.
260/280
The results are as follows:
Experiment Efficiency of WBCs RNA yield, A
260/280
capture on the filter, % ug/blood sample
First (embodiment of 96 12 2.03
the invention)
Second 74 8.9 2.10
This example demonstrates the improvement in leukocyte purification and total
RNA yield according to an embodiment of the invention.
EXAMPLE 4
This example demonstrates that leukocytes can be purified and lysed and the
leukocyte genomic DNA can be analyzed according to an embodiment of the invention.
Leukocytes are purified and recovered as described in Example 1.
200 μL aliquots of eluted leukocytes are processed to isolate genomic DNA using
phenol/chloroform extraction as is known in the art. The isolated DNA is analyzed via
UV-spectrophotometry, agarose gel electrophoresis, and PCR.
The results are as follows (wherein K=1000):
WBC Sample WBC concentration K/μL DNA yield μg/sample OD 260/280
1 2.95 2.9 1.94
2 2.65 2.5 1.98
3 3.00 3.1 1.82
Agarose gel electrophoresis of the obtained DNA shows high molecular weight
DNA is isolated from the recovered leukocytes. The DNA is intact and amplifiable, shown
by amplification using PCR and amelogenin primers.
EXAMPLE 5
This example demonstrates that leukocytes can be purified and lysed and the
leukocyte nucleic acids can be analyzed according to an embodiment of the invention, using
varied layers of leukocyte depletion media.
The filter devices are 25 mm Acrodisc® syringe filters with 4 or 8 layers of
Leukosorb leukocyte depletion media (Pall Corporation, Port Washington, NY).
9 mL samples of freshly collected blood from donors are treated with EDTA
anticoagulant, withdrawn into 10 μL syringes, and processed as follows. Influent and
effluent leukocyte counts are taken.
The inlet ports of the devices are connected to the filled syringes, the outlet ports
are connected to tubing leading to a waste container, the syringe plungers are pressed, and
samples of blood are passed at a flow rate of 3-5 drops per second through the filters. The
filtered blood is discarded.
New 5 mL syringe barrels (including plungers) are connected to the inlet ports of
the devices, and the tubing connected to the outlet ports of the devices are placed in waste
containers containing about 8 μL of phosphate buffered saline (PBS). The syringe plungers
are withdrawn to pull PBS from the containers, through the downstream surfaces of the
leukocyte depletion media and the upstream surfaces, and into the syringe barrels. After the
PBS is pulled into the syringe barrels, the syringe plungers are pulled out of the barrels, while
the device outlets remain in contact with the PBS in the waste containers. The PBS in each
syringe barrel then drains, via gravity through the upstream surface and the downstream
surface, and into a waste container. The wash is repeated, as another volume of PBS is pulled
through the media and allowed to drain as described above.
2.5 mL of pH-adjusted Lysis/Binding Solution is passed through the medium
using a syringe, and collected in a lysate collection tube. Lysate is added with 2.5 mL of
Nuclease-free Water, and Proteinase K. Total RNA is isolated as described in
“LeukoLOCK™ Total RNA Isolation System, Globin mRNA-Depleted Total RNA from
Whole Blood Samples,” Instruction Manual, Catalog #AM 1923, AM1933, AM1934,
(Manual 1923M Revision B) Ambion Inc., Austin, TX (2007) (the “LeukoLOCK™ Total
RNA Isolation System Manual”).
The efficiency of leukocyte capture, the RNA yield, and the optical density ratio
of absorbance at 260 and 280 nm (A ) is determined.
260/280
The results are as follows:
Filter device Efficiency of WBCs RNA yield, A
260/280
capture on the filter, % ug/blood sample
4 layers 67.2 9.5 2.06
8 layers 99.5 13.6 2.11
EXAMPLE 6
This example demonstrates the reduction in red blood cell and platelet
contamination while purifying and recovering leukocytes according to an embodiment of the
invention.
The system used for carrying out this Example is shown in Figure 2.
The filter devices are PURECELL® PXLA High Efficiency Leukocyte Reduction
Filters for Apheresis Platelet Transfusion with leukocyte depletion media (Pall Corporation,
Port Washington, NY). The inlet port 2 of the filter device 1 is connected through tubing and
a “Y” connector to the blood bag 4 and to the leukocyte sampling bag 9, the device outlet
port 3 is connected to tubing leading through another “Y“ connector to the waste bag 6 and to
the injection port 8, which is connected to syringe 7. The system is arranged horizontally on
a bench-top, with the waste bag hanging from the side of the bench.
The source bag, containing 50 mL of whole blood mixed with anticoagulant and
mL of air is placed on a box so that the bag is 2 inches above the filter on the bench-top.
The waste bag is hung below the bench-top to prevent reverse flow. The clamps are opened
allowing the blood to pass from the source bag through the filter and into the waste bag. The
tubing below the filter is clamped, and the filter backwashed via the injection port using PBS
or PBS/dextran solutions 6% dextran 70 in PBS, in 20 mL increments for a total wash
volume of 20, 40, or 60 mL, or in single 40 mL or 60 mL increments, and the wash fluid
passes into the source bag. For the control, the filter is not backwashed.
The wash fluid and cells are mixed in the source bag and the system is again
placed on the bench-top so that the source bag is 2 inches above the filter and the wash bag
hangs from the side of the bench. The mixture passes from the source bag through the filter
and into the waste bag.
The wash procedure is repeated until the specific wash volume is achieved.
The results are analyzed. No backwash yields red blood cell contamination of
about 14%, a 20 mL PBS backwash reduces red blood cell contamination to about 6.5%, a 2
x 20 mL PBS backwash reduces it to about 3.4%, and a 3 x 20 mL PBS backwash reduces it
to about 1.8%.
The leukocyte recovery without a backwash is about 61%, about 66% for 20 mL
and 2 x 20 mL PBS backwashes, and about 63% for a 3 x 20 mL PBS backwash.
No backwash yields platelet contamination of about 19%, as does a 20 mL PBS
backwash. A 2 x 20 mL PBS backwash reduces platelet contamination to about 13%, and a 3
x 20 mL PBS backwash reduces it to about 6%.
With respect to PBS/dextran, no backwash yields red blood cell contamination of
about 14%, about 69% leukocyte recovery, and about 22% platelet contamination.
The use of PBS/dextran reduces red blood cell contamination (about 8% 20 mL
PBS/dextran; about 3% for 2 x 20 mL PBS/dextran), and, platelet contamination (about 9%
mL PBS/dextran and 2 x 20 mL PBS/dextran). However, the use of PBS/dextran reduces
leukocyte recovery compared to no backwash (about 69% no backwash, 66% 20 mL
PBS/dextran; about 56% for 2 x 20 mL PBS/dextran).
In another experiment, comparing the results of a control, and PBS backwashes of
2 x 20 mL, 40 mL single backwash, and 60 mL single backwash, the results are generally
similar for the 3 backwashes (all showing increased leukocyte recovery, and lower red blood
cell and platelet contamination compared to the control), although the 2 x 20 mL backwash
yields about 18% red blood cell contamination, and the other backwashes yield about 8% red
blood cell contamination (compared to about 22% for the control).
EXAMPLE 7
This example demonstrates the reduction in DMSO concentration while
recovering leukocytes according to an embodiment of the invention.
Blood is collected from a donor into VACUTAINER® tubes (Becton Dickenson,
Franklin Lakes, NJ) containing EDTA anticoagulant. The blood is processed to obtain and
isolate buffy coat.
A cryoprotectant fluid containing 10% DMSO and 90% same donor plasma is
prepared, chilled, and mixed with the buffy coat. Two 6 mL samples of buffy coat mixed
with cryoprotectant fluid are obtained.
The samples are filtered, and the leukocytes are washed and eluted as generally
described in Example 1, with the exception that the leukocytes in this Example are eluted
with PBS, rather than an elution solution including dextran.
Influent, effluent, and eluted leukocyte cell counts are taken. Additionally, the
initial total protein concentration is determined, as is the total protein concentration in the
eluted leukocyte-containing fluid, as the change in protein concentration correlates with the
reduction of DMSO concentration. The results are as follows:
sample WBC recovery % Residual DMSO concentration %
1 65 0.11
2 62 0.14
The results show that DMSO concentration can be decreased by about 90% while
recovering leukocytes.
EXAMPLE 8
This example demonstrates leukocytes can be purified and recovered while
minimizing platelet contamination according to an embodiment of the invention.
Blood samples are filtered and leukocytes are washed and eluted using devices as
generally described in Example 1.
Influent, effluent, and eluted leukocyte and platelet counts are taken. The results
are as follows (wherein K=1000).
Sample Platelet Platelet Platelet count Reduction Residual WBC
ID count count in eluted in platelets Platelets in recovery in
influent, effluent, WBCs, K/μL on the eluted eluate %
K/μL K/μL filter, % WBC, %
Blood with EDTA anticoagulant
1 380 321 14
85 4 62
2 380 323 15
79 4 75
3 380 301 17
62 11 86
4 190 118 15
59 8 88
190 112 20
85 4 84
Blood with CP2D anticoagulant
1 335 21 4
6 1 63
2 335 168 24
50 7 84
3 335 157 17
47 5 80
4 175 34 7
19 4 74
175 49 9
28 5 78
6 175 60 8
34 5 72
EXAMPLE 9
This example demonstrates the reduction in red blood cell contamination while
purifying and recovering leukocytes and maintaining a closed system in according to an
embodiment of the invention.
The system 100 used for carrying out this Example is shown in Figure 3, showing
a filter device 1, having an inlet port 2 and an outlet port 3, source bag (blood bag) 4, waste
bag 6, leukocyte sampling bag 9, syringe 7 containing wash solution, syringe 8 containing
elution solution, and clamps 10-14.
The filter devices are PURECELL® PXLA High Efficiency Leukocyte Reduction
Filters for Apheresis Platelet Transfusion with leukocyte depletion media (Pall Corporation,
Port Washington, NY). The inlet port 2 of the filter device 1 is connected through tubing and
a “Y” connector to the blood bag 4 and to the leukocyte sampling bag 9, the device outlet
port 3 is connected via a “Y” connector to tubing leading to the waste bag 6, and to another
“Y” connector leading to syringes 7 and 8.
In order to control the flow, clamps 10-14 are placed on the tubing between the
inlet port and the blood bag and the sampling bag (clamps 10 and 11), and between the outlet
port and the waste bag and the wash and the elution syringes (clamps 12-14). All clamps are
initially closed.
The blood bag contains 50 mL of blood collected from donors and mixed with
anticoagulant according to a standard blood bank procedure. Clamps 10 and 13 are opened,
blood is filtered via gravity, passing into waste bag 6 and leucocytes are captured on the
leukocyte depletion medium in the filter device 1.
Clamp 13 is closed, the clamp 12 is opened and 20 mL of PBS is injected via
syringe 7 such that the wash solution passes through the downstream surface of the leukocyte
depletion medium and through the upstream surface and into blood bag 4. Clamp 12 is
closed and clamp 13 is opened again and wash solution is allowed to drain via gravity
through the filter device into the waste bag 6.
The wash procedure is repeated once more with fresh PBS. The wash fluid is
drained into a waste bag 6.
Clamps 10 and 13 are closed. Clamps 11 and 14 are opened and 24 μL of the
elution solution is injected from the syringe 8 such that leukocytes are eluted and pass, with
elution solution, into the sampling bag 9.
The results show, similar to those in Example 2, using the wash reduced red blood
cell contamination as compared to no wash. Additionally, this Example shows an
embodiment of the method can be carried out while maintaining a closed system.
All references, including publications, patent applications, and patents, cited
herein are hereby incorporated by reference to the same extent as if each reference were
individually and specifically indicated to be incorporated by reference and were set forth in
its entirety herein.
The use of the terms “a” and “an” and “the” and similar referents in the context of
describing the invention (especially in the context of the following claims) are to be
construed to cover both the singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. The terms “comprising,” “having,” “including,” and
“containing” are to be construed as open-ended terms (i.e., meaning “including, but not
limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely
intended to serve as a shorthand method of referring individually to each separate value
falling within the range, unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually recited herein. All methods
described herein can be performed in any suitable order unless otherwise indicated herein or
otherwise clearly contradicted by context. The use of any and all examples, or exemplary
language (e.g., “such as”) provided herein, is intended merely to better illuminate the
invention and does not pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best
mode known to the inventors for carrying out the invention. Variations of those preferred
embodiments may become apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventors expect skilled artisans to employ such variations as
appropriate, and the inventors intend for the invention to be practiced otherwise than as
specifically described herein. Accordingly, this invention includes all modifications and
equivalents of the subject matter recited in the claims appended hereto as permitted by
applicable law. Moreover, any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise indicated herein or
otherwise clearly contradicted by context.
In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as an admission that
such documents, or such sources of information, in any jurisdiction, are prior art, or form part
of the common general knowledge in the art.
In the description in this specification reference may be made to subject matter
which is not within the scope of the claims of the current application. That subject matter
should be readily identifiable by a person skilled in the art and may assist in putting into
practice the invention as defined in the claims of this application.
Claims (17)
1. A method for purifying leukocytes, the method comprising (a) passing a biological fluid comprising leukocytes, or a cryoprotectant fluid comprising leukocytes, through a porous leukocyte depletion medium having an upstream surface and a downstream surface, the fluid passing from the upstream surface through the downstream surface, wherein leukocytes are retained by the leukocyte depletion medium; (b) passing a wash fluid through the leukocyte depletion medium from the downstream surface through the upstream surface; and, (c) passing the wash fluid through the leukocyte depletion medium from the upstream surface through the downstream surface via gravity.
2. The method of claim 1, comprising passing the biological fluid or the cryoprotectant fluid through the porous leukocyte depletion medium via gravity
3. The method of claim 1, comprising passing the biological fluid or the cryoprotectant fluid through the porous leukocyte depletion medium via vacuum.
4. The method of any one of claims 1-3, further comprising repeating (b) and (c) at least once with additional wash fluid.
5. The method of any one of claims 1-4, wherein the cryoprotectant fluid comprises DMSO.
6. The method of any one of claims 1-5, wherein passing the wash fluid through the leukocyte depletion medium from the downstream surface through the upstream surface comprises creating a vacuum upstream of the upstream surface.
7. The method of any one of claims 1-5, wherein passing the wash fluid through the leukocyte depletion medium from the downstream surface through the upstream surface comprises creating a positive pressure downstream of the downstream surface.
8. The method of any one of claims 1-7, further comprising eluting leukocytes from the leukocyte depletion medium by passing an elution fluid through the leukocyte depletion medium from the downstream surface through the upstream surface, and recovering the eluted leukocytes.
9. The method of any one of claims 1-7, further comprising lysing the retained leukocytes.
10. The method of claim 9, further comprising processing nucleic acids from the lysed leukocytes.
11. The method of claim 10, further comprising purifying nucleic acids from the lysed leukocytes.
12. The method of claim 10 or 11, further comprising analyzing nucleic acids from the lysed leukocytes.
13. The method of claim 12, comprising determining the total RNA from the lysed leukocytes.
14. The method of claim 12, comprising depleting mRNA from the lysed leukocytes.
15. The method of any one of claims 1-14, carried out using a multiwell device, the device having a plurality of wells, each well having a porous leukocyte depletion medium therein, wherein separate portions of biological fluid and wash fluid are passed through the leukocyte depletion media in separate wells.
16. Leukocyte cells prepared by the method of claim 8.
17. A use of the leukocyte cells of claim 16 in the manufacture of a medicament.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/311,046 US20130143195A1 (en) | 2011-12-05 | 2011-12-05 | Leukocyte purification |
US13/311,046 | 2011-12-05 |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ603760A NZ603760A (en) | 2013-05-31 |
NZ603760B true NZ603760B (en) | 2013-09-03 |
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