US007150834B2
`
`(12) United States Patent
`Mueth et al.
`
`(10) Patent No.:
`
`(45) Date of Patent:
`
`US 7,150,834 B2
`Dec. 19,2006
`
`(54) MULTIPLE LAMINAR FLOW-BASED RATE
`ZONAL OR ISOPYCNIC SEPARATION WITH
`HOLOGRAPHIC OPTICAL TRAPPING OF
`BLOOD CELLS AND OTHER STATIC
`COMPONENTS
`
`(75)
`
`(73)
`
`Inventors: Daniel M. Mueth, Chicago, IL (US);
`Amy Anderson, Prospect Heights, IL
`(US); Jessica Shireman, Kansas City,
`MO (US)
`Arryx, Inc., Chicago, IL (US)
`
`Assignee:
`
`(*)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 54 days.
`
`(21)
`
`Appl. No.: 10/867,328
`
`(22)
`
`Filed:
`
`Jun. 13, 2004
`
`(65)
`
`Prior Publication Data
`
`US 2005/0061962 A1
`
`Mar. 24, 2005
`
`(63)
`
`(51)
`
`(52)
`
`(58)
`
`Related U.S. Application Data
`
`Continuation-in-part of application No. 10/630,904,
`filed on Jul. 31, 2003.
`
`Int. Cl.
`
`(2006.01)
`B01D 21/01
`U.S. Cl.
`.................... .. 210/732; 210/800; 210/802;
`210/927; 435/173.1; 422/72; 422/82.05; 422/101;
`436/177
`
`Field of Classification Search .............. .. 250/251;
`435/173.1; 210/732, 800, 802, 804, 927;
`436/177; 422/82.05, 72, 101
`See application file for complete search history.
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`............. .. 210/732
`4,409,106 A * 10/1983 Furuta et al.
`4,424,132 A *
`1/1984 Iriguchi
`.................... .. 210/800
`6,815,664 B1* 11/2004 Wang et al.
`.............. .. 250/251
`6,833,542 B1* 12/2004 Wang et al.
`.............. .. 250/251
`
`* cited by examiner
`
`Primary Examiner—Kiet T. Nguyen
`(74) Attorney, Agent, or Firm—Jean C. Edwards, Esq.;
`Akerman Senterfitt
`
`(57)
`
`ABSTRACT
`
`The invention provides a method and apparatus for separat-
`ing blood into components, may be expanded to include
`other types of cellular components, and can be combined
`with holographic optical manipulation or other forms of
`optical tweezing. One of the exemplary methods includes
`providing a first flow having a plurality of blood compo-
`nents; providing a second flow; contacting the first flow with
`the second flow to provide a first separation region; and
`differentially sedimenting a first blood cellular component of
`the plurality of blood components into the second flow while
`concurrently maintaining a second blood cellular component
`of the plurality of blood components in the first flow. The
`second flow having the first blood cellular component is then
`differentially removed from the first flow having the second
`blood cellular component. Holographic optical traps may
`also be utilized in conjunction with the various flows to
`move selected components from one flow to another, as part
`of or in addition to a separation stage.
`
`6 Claims, 7 Drawing Sheets
`
`PROVIDE A FIRST FLOII HAVIIIS A FLURALIIY OF CDIPUIEIITS
`
`PROVIDE A ECUID FLOH
`
`CONTACT TIE FIRST FLW IIITH TIE SECOND FLOW TO PROVIM A FIRST SHMHATION REGION
`
`DIFFEPEIITIALLV SEDIIIENT A FIRST CMPOIEHT OF A PLURALITV
`OF COMPONENTS FPOH TIE FIRST FLOII
`INTO THE SECOND FLOII
`
`CINCURENTLV HAIIITAIN A SECOND COMPONENT OF THE PLIITALITY
`OF CMPONENTS IN THE FIRST FLW
`
`OIFFERENTIALLT ITEIIIWE THE SECOID FLO)! HAVING THE FIRST
`COIIPOIBIT PRJH THE FIRST FLOII HAVING TIE SECOND OOIIPDNEHT
`an
`ancu
`
`zc:
`
`IDDITIOIIAL
`Sfl’ARATION?
`YES
`PROVIDE A THIRD FLW
`
`605
`
`BIO
`
`=nI5
`
`520
`
`525
`
`B30
`
`SID
`
`845
`
`CONTACT THE FIRST FLOII IIITH THE TIIIHJ Fm! TO PROVIDE A SECOND SEPARATION REGION
`an=-
`
`II
`L
`HANIPULATION OR SEPARATION?
`
`YES
`
`Exhibit No. 1()17
`
`PGR of U.S. Patent 8,933,395
`
`

`
`U.S. Patent
`
`Dec. 19,2006
`
`Sheet 1 of 7
`
`US 7,150,834 B2
`
`FIG.1
`
`

`
`U.S. Patent
`
`Dec. 19,2006
`
`Sheet 2 of 7
`
`US 7,150,834 B2
`
`2?
`
`F211;.
`
`
`
`
`
`HOLOGRAPHICOPTICALTRAPS
`
`

`
`U.S. Patent
`
`Dec. 19,2006
`
`Sheet 3 of 7
`
`US 7,150,834 B2
`
`

`
`
`
`
`
` \\\\m.....................--.................-- \\\\m....
`
`
`
`
`
`
`
`
`
`\\
`
`
`
`\\
`
`
`
`\\
`
`
`
`\\
`
`
`
`\\
`
`
`
`\\
`
`
`
`

`
`U.S. Patent
`
`Dec. 19,2006
`
`Sheet 5 of 7
`
`US 7,150,834 B2
`
`FIG. 5
`
`MICROSCOPE
`ILLUMINATION
`
`
`
`
`
`OPIICAL
`FIBER
`
`
`HOLOGRAPHIC
`OPTICAL IRAPPING
`/
`
`

`
`U.S. Patent
`
`Dec. 19,2006
`
`Sheet 6 of 7
`
`US 7,150,834 B2
`
`500
`
`FIG . BA
`
`PROVIDE A FIRST FLOW HAVING A PLUHALITY 0F COMPONENTS
`
`PROVIDE A SECOND FLOW
`
`“"5
`
`51°
`
`coNTAcT THE FIRST FLOW NITH THE SECOND FLOW To PROVIDE A FIRST SEPARATION REGION
`
`515
`
`53°
`
`525
`
`33”
`
`54”
`
`DIFFERENTIALLY SEDIMENT A FIRST COMPONENT OF A PLURALITY
`OF COMPONENTS FROM THE FIRST FLOW INTo THE SECOND FLOW
`
`CDNCURRENTLY MAINTAIN A SECOND COMPONENT OF THE PLURALITY
`OF COMPONENTS IN THE FIRST FLow
`
`DIFFERENTIALLY RENovE THE SECOND FLOW HAVING THE FIRST
`COMPONENT FRoN THE FIRST FLOW HAVING THE SECOND COMPONENT
`
`N0
`
`0
`
`835
`
`
`
`
`ADDITIONAL
`SEPARATION?
`
`
`
`YES
`
`PROVIDE A THIRD FLOW
`
`CONTACT THE FIRST FLOW NITH THE THIRD FLOW To PROVIDE A SECOND SEPARATION REGION
`
`545
`
`E50
`
`
`
`
`HOLOGHAPHIC
`
`HANIPULATION OR SEPARATION?
`
`
`

`
`U.S. Patent
`
`Dec. 19,2006
`
`Sheet 7 of 7
`
`US 7,150,834 B2
`
`FIG. 6B
`
`BB5
`
`DIFFERENTIALLY SEDIMENTING THE
`SECOND COMPONENT FROM THE FIRST
`FLOW INTO THE THIRD FLOW
`
`GENERATE A PLURALITY
`OF HOLOGRAPHIC TRAPS
`
`USING THE HOLOGRAPHIC TRAPS,
`DIFFERENTIALLY MOVING THE
`SECOND COMPONENT FROM THE FIRST
`
`FLOW INTO THE THIRD FLOW
`
`555
`
`550
`
`CONCURRENTLY MAINTAINING A THIRD COMPONENT OF THE PLURALITY
`OF COMPONENTS IN THE FIRST FLOW
`
`570
`
`DIFFERENTIALLY REMOVING THE THIRD FLOW HAVING THE SECOND
`COMPONENT FROM THE FIRST FLOW HAVING THE THIRD COMPONENT
`
`575
`
`RETURN
`
`880
`
`

`
`US 7,150,834 B2
`
`1
`MULTIPLE LAMINAR FLOW-BASED RATE
`ZONAL OR ISOPYCNIC SEPARATION WITH
`HOLOGRAPHIC OPTICAL TRAPPING OF
`BLOOD CELLS AND OTHER STATIC
`COMPONENTS
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`The present invention is a Continuation-In-Part Applica-
`tion of Lewis Gruber et al., U.S. patent application Ser. No.
`10/630,904, filed Jul. 31, 2003, entitled “SYSTEM AND
`METHOD OF SORTING MATERIALS USING HOLO-
`
`GRAPHIC LASER STEERING,” commonly assigned here-
`with, the contents of which are incorporated y reference
`herein, with priority claimed for all commonly disclosed
`subject matter (the “first related application”).
`
`FIELD OF THE INVENTION
`
`The present invention relates generally to techniques and
`systems for separation of cellular materials such as blood
`into its various cellular components and fractions, such as
`platelets, and more particularly, to a separation of blood or
`other biological materials into cellular components or other
`static components using multiple laminar flows and rate
`zonal or isopycnic separation, which further may be coupled
`with holographic optical trapping and manipulation.
`
`BACKGROUND OF THE INVENTION
`
`There are several categories of blood cells. Erythrocyte or
`red blood cell (RBC) counts are for women 4.8 million
`cells/ul and men 5.4 million cells/ul. RBCs make up 93% of
`the solid element in blood and about 42% of blood volume.
`
`Platelets are 2 p.m—3 um in size. They represent 7% of the
`solid elements in blood and about 3% of the blood volume,
`corresponding to about 1.5 to 4x10” cells per liter. There are
`5 general types of white blood cells (WBCs) or leukocytes
`accounting for about 1.5 to 4x109 cells per liter. The WBCs
`comprise: 50—70% Neutrophils (12—15 um in size); 241%
`Eosinophils (12—15 um in size); 0.5—1% Basophils (9—10
`um in size); 20410% Lymphocytes (25% B-cells and 75%
`T-cells) (8—10 um in size); and 3—8% Monocytes (16—20
`umin size). They comprise 0.16% of the solid elements in
`the blood, and approximately 0.1% of the blood volume
`corresponding to around 4 to 12><109 per liter. A subject with
`an infection might have a WBC count as high as 25><109 per
`liter.
`Platelets are the smallest cells in the blood and are
`
`important for releasing proteins into the blood that are
`involved in clotting. Patients with immune diseases that
`cause lower counts (such as cancer,
`leukemia and other
`chemotherapy patients) sometimes need platelet transfu-
`sions to prevent their counts from becoming too low. The
`platelet count in adults is normally between 140,000—440,
`000 cells/ul, and this number should not fall below 50,000
`cells/uL because platelets play an integral role in blood
`clotting.
`Blood separation techniques have traditionally employed
`discrete centrifugation processes. More particularly, a cer-
`tain volume of blood is removed from a donor at a particular
`time. That volume of blood is then subjected to different
`levels of centrifugation to provide corresponding blood
`fractions for blood components such as plasma, platelets, red
`blood cells, and white blood cells. This process is discrete,
`rather than continuous, such that if more blood from the
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`
`donor is to be processed, another volume is removed from
`the donor, and the process is repeated.
`The steps in platelet collection are: collection of blood
`from donor: addition of anticoagulant; separation via cen-
`trifugation; return of red cells, leukocytes and plasma to the
`donor. A collection normally contains about 20(L400 ml of
`plasma, which is reduced to avoid imcompatibility. This
`collection normally contains about 8 to 8.5><101° platelets. A
`donor normally gives approximately 10% of his/her platelets
`with no loss in clotting ability, although a larger number of
`platelets could be separated from the blood. These platelets
`must be used within five days of collection.
`Plateletpheresis, called apheresis,
`is a state of the art
`process by which platelets are separated [Haemonetics Com-
`ponent Collection System (CCS) and Multi Component
`System (Multi)(Haemonetics, Braintree, Mass.)]. This auto-
`mated machine separates platelets from blood over a period
`of 1.5 to 2 hours (assuming 10% donation). This process is
`faster than traditional approaches and is completely auto-
`mated and can be used for single or double platelet doses.
`Nevertheless, the process is slow relative to the patience of
`donors and is capable of improvement for the purity of the
`separated platelet fraction.
`Other procedures are also time consuming, often taking
`several hours, particularly when unused blood fractions are
`to be returned to the donor. For example, platelet donation
`make take several hours, as whole blood is removed from
`the donor, fractionated through centrifugation to obtain the
`platelets, and the remaining blood components are then
`injected back into the donor. This centrifugation process is
`also comparatively harsh, also can result in damage to a
`proportion of the harvested cells, effectively reducing the
`usable yield of the blood fractions.
`As a consequence, a need remains for a blood separation
`technique and apparatus which is continuous, has high
`throughput, provides time saving, and which causes negli-
`gible or minimal damage to the various blood components.
`In addition, such techniques should have further applicabil-
`ity to other biological or medical areas, such as for separa-
`tions of cellular, viral, cell organelle, globular structures,
`colloidal suspensions, and other biological materials.
`
`SUMMARY OF THE INVENTION
`
`invention
`The exemplary embodiments of the present
`provide for separating components in a mixture, such as
`separating the various blood components of whole blood
`into corresponding fractions, such as a platelet fraction, a red
`blood cell fraction, a white blood cell fraction, and a plasma
`fraction. The various embodiments of the present invention
`provide separation of components on a continuous basis,
`such as within a continuous, closed system, without the
`potential damage and contamination of prior art methods,
`particularly for fractionation of blood components. The
`continuous process of the present invention also provides
`significant time savings and higher throughput for blood
`fractionation. In addition, the various embodiments may also
`include additional means for separating and manipulating
`the components, particularly holographic optical manipula-
`tion and separation.
`An exemplary method of separating blood into compo-
`nents includes providing a first flow having a plurality of
`blood components; providing a second flow; contacting the
`first flow with the second flow to provide a first separation
`region; and differentially sedimenting a first blood cellular
`component of the plurality of blood components into the
`second flow while concurrently maintaining a second blood
`
`

`
`US 7,150,834 B2
`
`3
`cellular component of the plurality of blood components in
`the first flow. The second flow having the first blood cellular
`component is then differentially removed from the first flow
`having the second blood cellular component.
`The various sedimentation steps of the present invention
`may be rate zonal or isopycnic. In addition, the first flow and
`the second flow are substantially non-turbulent, and may
`also be substantially laminar.
`In a selected embodiment, the first blood cellular com-
`ponent is a plurality of red blood cells and a plurality of
`white blood cells, and the second blood cellular component
`is a plurality of platelets. For the first blood cellular com-
`ponent, the plurality of white blood cells may be holographi-
`cally separated from the plurality of red blood cells. Other
`holographic manipulations of the present invention include
`holographically removing a plurality of contaminants from
`the first flow, holographically separating biological debris
`from the first flow, and holographically separating a plurality
`of second blood cellular components from the first flow.
`Additional separation stages may also be included, with
`the exemplary method providing a third flow; contacting the
`first flow with the third flow to provide a second separation
`region; and differentially sedimenting the second blood
`cellular component of the plurality of blood components to
`sediment into the third flow while concurrently maintaining
`a third blood component of the plurality of blood compo-
`nents in the first flow. In selected embodiments, the second
`blood cellular component
`is a plurality of platelets and
`wherein the third blood component is plasma.
`A second exemplary method of separating a fluid mixture
`into constituent, non-motile components, in accordance with
`the present invention,
`includes: providing a substantially
`laminar first flow having the fluid mixture, the fluid mixture
`having a plurality of components, the plurality of compo-
`nents having a corresponding plurality of sedimentation
`rates; providing a substantially laminar second flow; con-
`tacting the first flow with the second flow to provide a first
`separation region, the first flow and the second flow having
`a substantially non-turbulent interface within the separation
`region; differentially sedimenting from the first flow a first
`component of the plurality of components into the second
`flow to form an enriched second flow and a depleted first
`flow, while concurrently maintaining a second component of
`the plurality of components in the first flow, the first com-
`ponent having a first sedimentation rate of the plurality of
`sedimentation rates and the second component having a
`second sedimentation rate of the plurality of sedimentation
`rates, wherein the first sedimentation rate is comparatively
`greater than the second sedimentation rate; differentially
`removing the enriched second flow from the depleted first
`flow; and holographically manipulating the second compo-
`nent in the depleted first flow.
`The second exemplary method may also include addi-
`tional separation stages, such as a holographic separation,
`including: providing a third flow; contacting the depleted
`first flow with the third flow to provide a second separation
`region; and holographically trapping the second component
`and moving the second component from the depleted first
`flow into the third flow while concurrently maintaining a
`third component of the plurality of components in the
`depleted first flow.
`An exemplary apparatus embodiment of the invention for
`separating a fluid mixture into constituent, non-motile com-
`ponents includes: a first sorting channel having a first inlet
`for a first flow and a second inlet for a second flow; the first
`sorting channel further having a first outlet for the first flow
`and a second outlet for the second flow, the first sorting
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`
`channel further having means to maintain the first flow and
`second flow substantially non-turbulent,
`the first sorting
`channel adapted to allow a first component in the first flow,
`of a plurality of components in the first flow, to sediment into
`the second flow to form an enriched second flow and a
`
`depleted first flow, while concurrently maintaining a second
`component of the plurality of components in the first flow;
`a second, optically transparent sorting charmel having a first
`optical inlet coupled to the first outlet for the first flow and
`having a first optical outlet, the second, optically transparent
`sorting channel further having a second optical inlet for a
`third flow and a second optical outlet for the third flow; and
`a holographic optical trap coupled to the second, optically
`transparent sorting charmel,
`the holographic optical
`trap
`adapted to generate a holographic optical trap to select and
`move the second component from the first flow into the third
`flow.
`
`Another apparatus or system for separating a plurality of
`components in a fluid comprises: an optically transparent
`sorting channel having a first inlet for a first flow and a
`second inlet for a second flow, the optically transparent
`sorting channel further having a first outlet for the first flow
`and a second outlet for the second flow; and a holographic
`optical
`trap system coupled to the optically transparent
`sorting channel, the holographic optical trap system adapted
`to generate a holographic optical trap to select and move a
`first component in the first flow, of a plurality of components
`in the first flow, into the second flow to form an enriched
`second flow and a depleted first
`flow, while a second
`component of the plurality of components is concurrently
`maintained in the first flow.
`
`Another method embodiment provides for separating a
`plurality of cells, comprising: providing a first flow having
`the plurality of cells; providing a second flow; contacting the
`first flow with the second flow to provide a first separation
`region; and differentially sedimenting a first cell of the
`plurality of cells into the second flow while concurrently
`maintaining a second cell of the plurality of cells in the first
`flow. The method generally also includes differentially
`removing the second flow having the first cell from the first
`flow having the second cell. The method may also provide
`for providing a third flow; contacting the first flow with the
`third flow to provide a second separation region; and dif-
`ferentially sedimenting the second cell of the plurality of
`cells into the third flow while concurrently maintaining a
`third cell of the plurality of cells in the first flow. In addition,
`a plurality of second cells may be holographically separated
`from the first
`flow, and a plurality of contaminants or
`biological debris may be holographically removed from the
`first flow.
`
`Numerous other advantages and features of the present
`invention will become readily apparent from the following
`detailed description of the invention and the embodiments
`thereof, from the claims and from the accompanying draw-
`ings.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`The objects, features and advantages of the present inven-
`tion will be more readily appreciated upon reference to the
`following disclosure when considered in conjunction with
`the accompanying drawings, in which:
`FIG. 1 is a lateral view of an apparatus 100 in accordance
`with the present invention.
`FIG. 2 is an illustration of optical trapping for component
`separation in an apparatus 200.
`
`

`
`US 7,150,834 B2
`
`5
`FIG. 3 is a diagram illustrating a closed, two-stage system
`300 for blood component separation in accordance with the
`present invention.
`FIG. 4 schematically illustrates a holographic optical
`trapping system according to one embodiment consistent
`with the present invention.
`FIG. 5 is a schematic diagram of a holographic optical
`trapping system for sorting objects according to one embodi-
`ment in accordance with the present invention.
`FIG. 6 (divided into FIG. 6A and FIG. 6B) is a flow
`diagram illustrating a method embodiment of the present
`invention.
`
`DETAILED DESCRIPTION OF THE
`EXEMPLARY EMBODIMENTS
`
`While the present invention is susceptible of embodiment
`in many different forms, there are shown in the drawings and
`will be described herein in detail specific embodiments
`thereof, with the understanding that the present disclosure is
`to be considered as an exemplification of the principles of
`the invention and is not intended to limit the invention to the
`
`specific embodiments illustrated.
`As indicated above,
`the various embodiments of the
`present invention provide for separating components in a
`mixture, such as separating the various blood components of
`whole blood into corresponding fractions, such as a platelet
`fraction, a red blood cell fraction, a white blood cell fraction,
`and a plasma fraction. The various embodiments, as
`described below, utilize one or more sorting channels,
`having a plurality of substantially laminar flows, allowing
`one or more components to differentially sediment from one
`flow into another, thereby separating the components into
`corresponding flows. In addition, the various components
`may be sorted further using optical mechanisms, such as
`holographic optical trapping. The various embodiments of
`the present invention thereby provide separation of compo-
`nents on a continuous basis, such as within a continuous,
`closed system, without the potential damage and contami-
`nation of prior art methods, particularly for fractionation of
`blood components. The continuous process of the present
`invention also provides significant time savings for blood
`fractionation.
`
`In addition to whole blood sorting and fractionation
`applications, the present invention is also suitable for other
`cell sorting applications, such as separations of cancer cells
`from normal or healthy cells in, for example, bone marrow
`extractions. The various embodiments of the present inven-
`tion have further applicability to other biological or medical
`areas, such as for separations of cells, viruses, bacteria,
`cellular organelles or subparts, globular structures, colloidal
`suspensions,
`lipids and lipid globules, gels,
`immiscible
`particles, blastomeres, aggregations of cells, microorgan-
`isms, and other biological materials. For example, the com-
`ponent separation in accordance with the present invention
`may include cell “washing”, in which contaminants (such as
`bacteria) are removed from cellular suspensions, which may
`be particularly useful in medical and food industry applica-
`tions. Significantly, prior art flow-based techniques have not
`recognized any applicability to sorting or separation of
`non-motile cellular components using variable sedimenta-
`tion rates and optical manipulation.
`FIG. 1 is an illustration of a lateral view of an apparatus
`100 in accordance with the present invention. As illustrated
`in FIG. 1,
`the sorting apparatus 100 includes a sorting
`channel 110, a plurality of inlets 120 and a plurality of
`outlets 130. A corresponding fluid flow, such as illustrated
`
`6
`flows W, X, Y and Z, enters one of the inlets 120 and flows,
`substantially non-turbulently or otherwise as a laminar flow,
`across the sorting charmel (or sorting region) 110, and out
`through a corresponding outlet 130, as illustrated.
`The apparatus 100 (and 200, below) may be constructed
`of a plurality of materials, integrally or as discrete compo-
`nents, using a wide variety of materials, such as metals,
`ceramics, glass, and plastics. In selected embodiments, when
`coupled with holographic trapping or other form of optical
`tweezing, the apparatus 100 (or 200) is transparent to the
`selected wavelength of the holographic generator, such as
`optically transparent when the holographic generator utilizes
`visible wavelengths. Depending upon the selected applica-
`tion, the apparatus 100 (200) should also be sterile and may
`also have a controlled temperature. The various fluid flows
`may be fed into the inlets 120 through a wide variety of
`means known to those of skill in the art and are within the
`
`10
`
`15
`
`scope of the present invention, including use of peristaltic
`pumps or a gravity feed, for example, and such means may
`also be utilized to control the flow rates of the various flows
`
`20
`
`to
`W, X, Y and Z. When peristaltic pumps are utilized,
`maintain a constant flow rate and pressure, bubble-traps may
`be incorporated at the inlets 120 of the apparatus 100 (or
`200).
`The various fluids utilized in the separation flows may be
`diluted or concentrated, to increase or decrease the volume
`of one of the solutions, or to impact the concentration of
`some dissolved or suspended material, or to impact physical
`properties of the solution such as its viscocity, temperature,
`or density. Examples for the apparatus 100, when used for
`blood sorting, include: (a) dilution of the blood to reduce
`clogging or hydrodynamic interaction between blood cells,
`(b) extension of the volume of the blood or a blood fraction,
`(c) modification of the density of the blood, a blood fraction,
`or another solution which impacts the flow properties or
`separation behavior, (d) extension of the volume of a solu-
`tion to maintain in increase fluid volume, especially in
`circumstances when fluid volume is being removed from the
`system.
`The various fluids utilized in the separation flows also
`may be “activated”, such that some process is activated
`within the solution by some external influence or mixing
`with an external solution. Examples of external influences
`include: (a) applying an electric field, (b) applying a mag-
`netic field, (c) exposing to light, (d) modifying the tempera-
`ture, (e) introducing a chemical, (f) introducing a biological
`material, (g) shearing the solution, and (h) vibrating the
`solution. Examples of the activation which is caused by the
`external solution include: (a) alignment of particles, mol-
`ecules, or cells, (b) polarization of one or more components
`of the solution, (c) cross-linking, (d) initiation or termination
`of chemical reaction,
`(e)
`initiation or termination of a
`biological response,
`(f) changing the type or rate of a
`chemical, physical, or biological response, or (g) causing a
`response or separation which depends upon the character of
`the particular component which is responding. Examples for
`the apparatus 100, when used for blood sorting, include: (a)
`addition of an agent to reduce clotting; (b) addition of agents
`to preserve viability or health of the blood solution or its
`components; (c) addition of agents which may augment the
`sorting process, such as by binding or collecting near certain
`components, thereby influencing one or more of their physi-
`cal properties,
`including the addition of beads or other
`particles or polymers which may adhere to one or more
`species, and also including the introduction of salts or other
`materials which may influence the electrostatic interaction
`of materials or the hydrodynamic size or character of the
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`

`
`US 7,150,834 B2
`
`7
`materials; (d) addition of agents which may influence the
`flow properties, such as by changing the density, viscosity,
`surface tension, or other parameters; (e) addition of agents
`to enhance or suppress the aggregation of certain materials;
`and (f) addition of agents to enhance or suppress the
`adherence of certain materials to other materials or parts of
`the flow device.
`In accordance with the invention, one of the fluid flows,
`such as the illustrated flow W, contains a plurality of
`components A, B, C and E. For example, when the fluid is
`whole blood,
`these components may be red blood cells
`(“RBC”), white blood cells (“WBC”), platelets, cellular
`debris and contaminants, all in plasma. Typically, many of
`the plurality of components have different sedimentation
`rates, typically measured using a Svedberg coeflicient. For
`example, RBCs have a comparatively greater sedimentation
`rate than platelets, and will be expected to sediment faster on
`a passive basis, such as due to gravitational or buoyant
`forces, without the intervention of other, active mecham sms,
`such as centrifugation. As the various flows W, X, Y and Z
`flow through the sorting region 110, based upon different
`sedimentation rates, the plurality of components (such as
`cells or other particles) will sediment, moving from one flow
`to another. As illustrated, component A having the compara-
`tively greatest sedimentation rate is illustrated as having
`moved from flow W to the lowest flow Z, component B
`having the comparatively next highest sedimentation rate is
`illustrated as having moved from flow W to the flow Y
`(above Z), component C having a comparatively smaller
`sedimentation rate is illustrated as having moved from flow
`W to the flow X (above Y), while component E having the
`comparatively smallest sedimentation rate, is illustrated as
`having remained in flow W (above Y). Using these different
`sedimentation properties, each of these components may be
`separated into a corresponding flow, and isolated from each
`other as each flow exits through its corresponding outlet 130.
`As each flow W, X, Y and Z exits through its corresponding
`outlet 130, that flow is differentially removed from the other
`flows, i.e., the flow is removed while the other flow remains
`intact or is otherwise separately removed from the remaining
`flows. In addition, this differential removal may be concur-
`rent, namely, all flows removed concurrently or continu-
`ously.
`Continuing to refer to FIG. 1, using whole blood with an
`anticoagulant (such as sodium citrate or heparin) as the fluid
`flow W, for example, the various blood fractions may be
`separated from each other, with red blood cells sedimenting
`fastest and represented by component A (e.g., 4.59 um/s),
`white blood cells sedimenting at a slightly lower rate and
`represented by component B (e.g., 2.28 um/s), platelets
`sedimenting at a comparatively slower rate and represented
`by component C (e.g., 0.055 um/s), and plasma continuing
`to comprise flow W and represented by component E. Each
`blood fraction may then be removed through a correspond-
`ing outlet 130.
`Not separately illustrated in FIG. 1, due to buoyant forces
`and relative density considerations, there may be particles or
`components in one or more of the fluid flows which will flow
`up to a higher flow (e.g., creaming). For example, less dense
`particles entering through flow X may rise into flow W, and
`exit with flow W through a corresponding outlet 130.
`Illustrated in lateral view, the sorting channel (or sorting
`region) 110 of apparatus 11 has a varied length “L” parallel
`to the direction of flow, a depth “D” perpendicular to the
`direction of flow, and a width (not illustrated, extending into
`the page). These various dimensions may be selected based
`on a plurality of factors, particularly the flow rates and the
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`8
`sedimentation rates of the components of interest. For
`example, for a selected flow rate, the total length of the
`sorting charmel should be long enough to differentially
`remove the component having the comparatively slowest
`sedimentation rate, illustrated as component C in FIG. 2,
`with shorter lengths corresponding to other flows for sepa-
`ration of components having faster sedimentation rates, as
`illustrated for flow Z having component A and flow Y having
`component B.
`Flow rates may also vary between the plurality of flows
`utilized in apparatus 100. For example, higher flow rates in
`the lower level flows (such as Y and Z) may tend to
`compress the flows W and X, resulting in a shorter distance
`that certain components must traverse to sediment into the
`flows Y and Z.
`
`In addition, the sedimentation of components through the
`various flows of the apparatus 100 is typically rate zonal,
`that is, based upon both relative density and size of the
`components to be separated, as well as the material’s shape
`and electrostatic properties. Under other conditions, how-
`ever, such as slower flow rates, thinner flow depths, and/or
`longer sorting channels 100, the sedimentation may also be
`isopycnic, that is, based only upon relative density of the
`components. When isopycnic separation is desired,
`the
`various fluids comprising the flows W, X, Y and Z may be
`selected and adjusted to create desired density gradients to
`match the component densities for the selected separations.
`The various fluids comprising the generally laminar
`flows, such as flows W, X, Y and Z, may also be selected
`based on suitable criteria for the particular desired compo-
`nent separation. For example, for blood separation,
`the
`various flows may be comprised of whole blood, such as for
`flow W, and