WO2013085797A1

WO2013085797A1 - Method of washing cells using passive separation of a lysed blood sample with clean buffer using inertial microfluidic separation and focusing in spiral microchannels

Info

Publication number: WO2013085797A1
Application number: PCT/US2012/067097
Authority: WO
Inventors: Robert W. APPLEGATE, Jr.
Original assignee: Focus Biomedical, Llc
Priority date: 2011-12-06
Filing date: 2012-11-29
Publication date: 2013-06-13

Abstract

A method and apparatus for the automatic, manual-free washing of stained, lysed cells as an anterior step to delivering the washed cell stream to a flow cytometer for subsequent counting and analysis is disclosed, which utilizes a passive, automatic, self-separating, inertial microfluidic flow in microchannels, whereby the washed cells are passively separated from a lysed solution and focused to the center microchannel of the device.

Description

METHOD OF WASHING CELLS USING PASSIVE SEPARATION OF A LYSED BLOOD SAMPLE WITH CLEAN BUFFER USING INERTIAL MICROFLUIDIC SEPARATION AND FOCUSING IN SPIRAL MICROCHANNELS

BACKGROUND OF THE INVENTION

The present invention is directed to the washing, or cleaning, of a lysed blood sample for subsequent delivery to a flow cytometer apparatus , where it is measured and analyzed.

Flow cytometry is used in research and clinical diagnostic settings for applications that include: AIDS progression tracking through CD4+ testing, high- throughput drug discovery, cell sorting, and many immunophentotyping assays for clinical diagnostics. Traditional flow cytometers hyrdodynamically focus a sample stream using a sheath fluid, which precisely aligns particles within a tightly focused sample stream that is queried using a focused laser beam. The intersection of the laser with the stream creates a small optical interrogation volume typically just larger than that of a single cell (a few picoliters). This tightly constrained optical interrogation volume enables flow cytometers to perform largely homogeneous assays and, as linear velocities of the focused particles can approach 10 m/s, allows sample analysis rates as high as 50,000 events per second. However, the use of hydrodynamic focusing requires significant volumes of a pure sheath fluid, and the high linear velocities generated create the need for very low-noise laser sources, highly sensitive detectors, and high-speed data acquisition systems. All of which, increase the cost, use of consumables, power consumption, and the size of traditional flow cytometers.

For these reasons, there have been many efforts to replace hydrodynamic focusing, while retaining the benefits (homogeneous assays, population analyses, simple optical interrogation) of analyzing a precisely positioned stream of flowing particles. Such efforts have led to the exploration of methods to focus particles without increasing their linear velocity, using acoustic forces, dielectrophoresis, and optical forces. These approaches require external energy, beyond that used to generate the fluid flow, to move particles to desired locations. Nonethess, they concentrate particles within the sample to a precise stream without a concurrent increase in linear velocity. Alternatively, microfabricated shape induced micro vortices, expansion channels, and inertial focusing have been shown to align cells and microparticles into desired streamlines, without external power requirements. In the case of the expansion channels, FITC labelled antibodies and FITC -doped silica nanospheres were used to perform a simple two parameter CD4 counting assay. In this study, measurements were based only on FITC intensities and optical precision of the instrument for general flow cytometry use was not evaluated.

Flow cytometry apparatuses are employed for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them through an electronic detection apparatus. Flow cytometry is routinely used in the diagnosis of health disorders, but has many other applications in both research and clinical practice. As a prerequisite, the blood sample from which the microscopic particles are to be counted must first be treated in order to provide a clean or washed solution containing the particles to be analyzed. This is typically accomplished by placing the lysed and stained blood sample in a centrifuge, which separates out the target cells from the lysed solution. The cells are then re-suspended in a clean buffer , after which the washed cells are delivered to the flow cytometer for analysis.

Hithertofore, this process of first collecting the blood sample, lysing it with a lysing buffer to separate out the red blood cells from the white blood cells, staining the white blood cells with the appropriate stain for labeling the type or types of white blood cells being assayed, and the spinning thereof in a centrifuge to remove the labeled white blood cells from the lysis solution and stain, and the re-suspension of the separated white cells in a clean buffer, have typically been done manually by one or more technicians. Advancements have been made whereby this process has been automated. An example of such an automatic apparatus is that manufactured by Copley Scientific' s M5000 Flow Cyto Prep, which interfaces with a flow cytometer, is a self-contained , bench-top instrument allowing for easy transport and set-up in a laboratory or field, and is capable of use for preparing clean samples for cell concentration, DNA, cell-cycle and size-distribution analysis, apoptosis measurements, immunofluorescence staining, managing and controlling bioreactors, as well as other protocols. The microchamber where the separation and washing occur utilizes a semi-permeable membrane for achieving the separation of the assayed cells. Details on this microchamber are disclosed in U.S. Patent 6,555,360. An example of another automated device for sample preparation for a flow cytometer is that manufactured by Becton Dickinson's FACS Lyse Wash Assistant (LWA). In this device, the separation and washing of the lysed cells is achieved in a microchamber, which microchamber separates the lysed cells through via a centrifuge or vortexer within the microchamber. Another example is the Beckman Coulter Q-Prep or Immunoprep apparatus.

Reference is also had to the published article entitled "Sample Preparation Using a Centrifuge-on-a-Chip", by Albert J. Mach, Jae Hyun Kim, Armin Arshi, Soojung Claire Hur and Dino Di Carlo, dated November, 2011.

In the field of the microfluidic flow, it has been determined that, under certain conditions of a specific range of Reynolds Number and Dean's Flow, particle focusing is achieved. The Dean Number reflects the vortex or turbulent flow at the boundaries of the otherwise, mainly laminar flow of the flow-stream. Choosing the appropriate channel-shape assists in this particle focusing. This inherent focusing causes the targeted micro-particles to flow along the center of the passageway, in what is called a first, center microchannel, while the remainder of the flowing fluid travels along the boundaries of the passageway in what are termed boundary or terminal microchannels. This phenomenon has been termed inertial focusing in microchannels. Based on experimental studies, it has been determined that curved passageways that thus provide curved microchannels achieve the most effective inertial focusing, based on the associated Reynolds Number and Dean's Flow characteristics. Curvilinear geometry of a spiral microchannel introduces a centrifugal acceleration component directed radially-outwardly, resulting in the symmetric-counter rotating vertices known as Dean' s vortices in the top and bottom halves of the microchannel. The magnitude of these Dean vortices may be as the Dean Number (De) which is

p is the density of the fluid, μ is the dynamic viscosity, V is the axial velocity scale, D is the diameter, R is the radius of curvature of the path of the channel. The Dean number is therefore the product of the Reynolds number (based on axial flow V through a pipe of diameter D) and the square root of the curvature ratio. For microdynamic flow in microchannels, two Reynolds Numbers are used and are defined as: R_c for unperturbed or laminar channel flow, and R_p for perturbed particle flow which includes data specific to the shape of the channel and the particles flowing therethrough, where R_r=^um ^Dh / v , and R_p = ^um ⁽a²) / vD, where U_m maximum channel velocity, v being the dynamic viscosity and density of the fluid, and D_h being the hydraulic diameter. For a straight microchannel, De is 0. Particles suspended in a spiral microchannel experiences transverse forces due to these Dean vortices.

U.S. Published Patent Application Number 2009/0014360 discloses a number of different systems and methods for using inertial microfluidic flow for the separating and focusing of particles, with a number of differently-shaped passageways disclosed and defining various Reynolds and Dean Numbers for achieving such focusing.

In the following published papers, this inertial focusing in microchannels has been amply explained: "Continuous Inertial Focusing, Ordering and Separation of Particles in Microchannels, by Dino Di Carlo, et al., dated November 27, 2007; "Inertial Microfluidics", by Dino Di Carlo, September 22, 2009; "Inertial Microfluidics for Continuous Particle Separation in Spiral Microchannels", by Sathyakumar S. Kuntaegowdanahalli, et al., July 21, 2009. All of these articles adequately explain the principals and physics behind inertial-flow focusing.

The concept of using inertial flow focusing in microchannels for a flow cytometer has, recently, also been known. In an article entitled "Particle Focusing in Staged Inertial Microfluidic Devices for Flow Cytometry", by John Oakey, Robert W. Applegate, Jr., et al., there is disclosed a method of focusing the cells and microparticles into a narrow sample stream utilizing microfluidic inertial focusing in the manner described above, instead of the usual and conventional method of hydrodynamically focusing the cells, or the use of other conventional focusing methods, such as acoustic, optical, and dielectrophoretic focusing. The microfluidic focusing occurs in a straight section of a flow-passageway directing the separated cells past the laser of the flow cytometer, with the actual cell separation occurring in an anterior curved section, such as a spiral passageway where the Dean Flow vortices and lift forces act on the flowing sample, as described hereinabove. The benefit of such inertial microfluidic focusing and separation in a flow cytometer is that the need for a liquid sheath when using hydrodynamic focusing is obviated. SUMMARY OF THE INVENTION

It is the primary objective of the present invention to provide a method and apparatus for the automatic, manual-free washing of stained, lysed cells or particles as an anterior step to delivering the washed cell stream to a flow cytometer for subsequent counting and analysis.

It is, also, the primary objective of the present invention to provide such a method and apparatus for the automatic, manual-free washing of stained, lysed cells using a passive, automatic, self-separating, inertial microfluidic flow in microchannels, whereby the washed cells or particles are passively separated from a lysed solution and focused to the center microchannel of the device.

It is, also, another primary objective of the present invention to provide such a method and apparatus for the automatic, manual-free washing of stained, lysed cells or particles, where the washed cells are passively separated from a lysed solution and focused to the center microchannel of the device utilizing a spiral passageway of N amount of spiral arms or turns, Ni amount of inlet or incoming turns, N₂ amount of turns of outlet or outgoing turns, where Ni may be, may not be, equal to N_2.

Toward these and other ends, the device of the invention first receives or draws in whole blood, and the mixes it with the correct amount of cell stain or stains for the particular assay being done or selected, the device 10 offering a number of different stains, lysis solutions, and reagents stored in canisters or storage containers for dispensing via pumps. The device then waits the correct incubation time, and then adds lysis buffer to the solution, and then awaits again a predetermined amount of time for the red-cell lysis to occur. After the lysing of the red cells has been completed, the device then pumps the solution and a clean buffer through the chip or cartridge , which chip constitutes the wash station where the stained cells are washed via the clean buffer passing through the center channel of the spiral microchannel passageway thereof, into a clean or washed stream of particles of white blood cells, and separated from the remainder of the blood, or red blood cells , and lysis buffer and any reagents flowing in the outer portion of the microchannel of the passageway. The separated waste is sent to a waste reservoir for subsequent safe disposal. The chip is inserted into the device at a slot or opening to which location the stained and lysed red blood cells and the clean buffer mix and enter for subsequent inertial separation. The chip is thus removable and replaceable with another chip, which replacement chip may have a passageway defining a spiral inertial microfluidic, microchannel flow channels different from that of the first and replaced chip, in order to accommodate a wide range of assay and environments, where each chip defines a unique inertial microfluidic, microchannel flow passageway configuration that is best suited to that particular assay or environment.

BRIEF DESCRIPTION OF THE DRAWING

The invention will be more readily understood with reference to the accompanying drawings, wherein:

Figure 1 is a diagrammatic view of the device of the present invention for accepting and treating whole blood until the final stage or step of delivering the stained and lysed solution to the chip or cartridge of the invention for washing a streaming flow of lysed cells, for subsequent delivery to a flow cytometer;

Figure 2 is a diagrammatic view of the device of the present invention for accepting and treating already-stained blood previously assayed with a specific and chosen assay, upon which it is mixed with a lysis buffer for subsequent delivery to the chip of the invention for washing at the final stage or step of washing a streaming flow of lysed cells, for subsequent delivery to a flow cytometer;

Figure 3 is a diagrammatic view of the device of the present invention for accepting and treating already- stained and lysed blood previously stained and assayed with a specific and chosen assay, for delivery to the chip of the invention for washing at the final stage or step of washing a streaming flow of lysed cells, for subsequent delivery to a flow cytometer;

Figure 4 is a flow chart listing the steps the software of the device of the invention performs in the device of Figure 1 ;

Figure 5 is a flow chart listing the steps the software of the device of the invention performs in the device of Figure 2;

Figure 6 is a flow chart listing the steps the software of the device of the invention performs in the device of Figure 3;

Figure 7 is a schematic view showing the spiral shape of the passageway in the interior of the chip or cartridge of the invention for use in the device of Figs. 1- 3, for focusing the washed cell stream to the center microchannel of the passageway;

Figure 8 is a diagram showing the flow stream at the input to the spiral channel of Fig. 7; and Figure 9 is a diagram showing the flow stream at the output of the spiral channel of Fig. 7, with the washed cell stream focused and separated from the remainder of the inlet-flow stream.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the drawings in greater detail, there is shown in Figs. 1, a diagram of the apparatus 10 of the invention that automatically performs what have hithertofore been done manually in the assaying and processing of blood samples. The apparatus 10 includes a main frame 12 in which are provided a number of stations for performing the relevant and necessary steps in the processing of the blood. Conventional pumps and valves are provided toward this end, as well as conventional storage containers for storing and dispensing necessary stains for staining the white blood cells to be separated out and analyzed in a flow cytometer, relevant assays for the particular white blood cell being analyzed, and any reagents, and a clean buffer solution. Cell lysis solutions are detergent-based, buffers and reagent sets that have been optimized for particular cell lysis applications. Effective cell lysis and protein extraction for different species of organisms and different cell and tissue types require different buffer formulations.

The apparatus 10 is an integrated system for preparing samples for flow cytometry in clinical, research, or forensic laboratory settings. The heart of the apparatus 10 is a chip or cartridge described hereinbelow in detail, which incorporates a spiral passageway having a linear inlet and outlet, which provide microchannels for the inertial, microdynamic-flow focusing and separation. The chip or cartridge is removably inserted into the apparatus, and, for purposes of scale, is about the size of a standard credit card. The apparatus 10 holds or stores lysis buffer, clean buffer, and all appropriate reagents within the device. Thus, the apparatus allows for the efficiency of a plug-and-play system even for lab situations that run a variety of tests using a variety of stains, and where one may substitute one cartridge for another having different microchannel-characteristics to suit the assay or test being done. The cartridge incorporates an entirely passive wash step, allowing for much higher quality data, less work by the technicians, and less human intervention, ultimately saving cost and consumables.

It is to be understood that above-mentioned automatic operation may be accomplished in a conventional manner, and may use or adopt that which is used in the automated device for sample preparation for a flow cytometer as that manufactured by Becton Dickinson's FACS Lyse Wash Assistant (LWA), as described hereinabove, except for the wash station which in accordance with the present invention utilizes inertial microfluidic, microchannel flow for focusing and separation of the washed stained white-blood cells particle stream from the lysed red blood cells, as described hereinbelow in detail.

The device 10 first receives or draws in whole blood, and the mixes it with the correct amount of cell stain or stains for the particular assay being done or selected, the device 10 offering a number of different stains, lysis solutions, and reagents stored in canisters or storage containers for dispensing via pumps. The device then waits the correct incubation time, and then adds lysis buffer to the solution, and then awaits again a predetermined amount of time for the red-cell lysis to occur. After the lysing of the red cells has been completed, the device then pumps the solution and a clean buffer through the chip or cartridge 20, which chip 20 constitutes the wash station where the stained cells are washed via the clean buffer, into a clean or washed stream of particles of white blood cells, and separated from the remainder of the blood, or red blood cells , and lysis buffer and any reagents. The chip 20 incorporates therein the above-mentioned inertial microfluidic, microchannel passageway , described hereinbelow in greater detail. The separated waste is sent to a waste reservoir 22 for subsequent safe disposal. The chip 20 is inserted into the device 10 at a slot or opening (not shown) to which location the stained and lysed red blood cells and the clean buffer solution enter for subsequent inertial separation. The chip 20 is thus removable and replaceable with another chip, which replacement chip may have a passageway defining an inertial microfluidic, microchannel flow channels different from that of the first and replaced chip 20, in order to accommodate a wide range of assay and environments, where each chip defines a unique inertial microfluidic, microchannel flow passageway configuration that is best suited to that particular assay or environment.

Referring to Fig. 2, there is shown the apparatus 10 used as a partially- automated device, where the input to the apparatus 10 is already stained blood samples. The device 10 draws or pumps in the stained blood into the device, and then mixes it with lysis buffer to remove the red blood cells. After a waiting period for assuring lysis, the device pumps the solution and clean or washing buffer through the chip 20, which chip 20 constitutes the wash station where the stained cells are washed via the clean buffer into clean or washed stream of particles of white blood cells, and separated from the remainder of the blood, or red blood cells , and lysis buffer and any reagents, as described above with reference to Fig. 1.

Referring to Fig. 3, there is shown the apparatus 10 where another partial automation is employed, and where the input to the apparatus 10 is already stained and lysed blood samples. The device 10 draws or pumps in the stained and lysed blood into the device, as well as the clean or washing buffer through the chip 20, which chip 20 constitutes the wash station where the stained cells are washed via the clean buffer into clean or washed stream of particles of white blood cells, and separated from the remainder of the blood, or red blood cells , and lysis buffer and any reagents, as described above with reference to Fig. 1.

In the device 10, a microcontroller 24 automatically controls the operation thereof, including the pumps, which may piezo pumps, valves, timing cycles, and the like, in a manner similar to the automated devices for sample preparation for a flow cytometer that is manufactured by Becton Dickinson's FACS Lyse Wash Assistant (LWA), or the Beckman Coulter Q-Prep or Immunoprep apparatus, described hereinabove.

Referring to Fig. 4, there are shown the method steps controlled by the microcontroller 24 for the device of Fig. 1. One first manually inserts the wash- station chip, or cartridge, 20 into the slot therefore (block 32), as well as the whole blood sample and then selects and activates a specific and chosen assay to be done on the blood sample. The microcontroller 24 senses the step in block 30, and then actuates a pump that sucks in the blood sample from its vial and adds the correct amount of relevant stains based on the assay selected, from reservoirs therefor, and then pumps the stained sample through a sample loop which allows for the requisite incubation time to occur within the tube (Block 32). Thereafter, the microcontroller controls pumps that pump the stained sample from the tube and the correct amount of lysis buffer from a storage reservoir thereof, and then pumps it through another sample loop to allow enough time for incubation within the tube to occur (block 34.) The microcontroller 24 then actuates a pump that pumps the stained and lysed blood sample through the outside microchannels of the passageway of chip 20, and also pumps a clean buffer solution from its respective reservoir and delivers them to the chip for flow through the center microchannel of the passageway of the chip 20 (blocks 36,38). The chip will then passively and centrally focus the stained white cells into the clean buffer stream (block 40), whereupon the washed lysed cells are exported to a vial for subsequent input into to a flow cytometer (block 42), while the waste is delivered to a waste-storage receptacle or reservoir (block 44).

In Fig. 5, the flow chart for the operation of the microcontroller 24 of the device of Fig. 2 is shown. Initially, one first manually inserts the wash-station chip 20 into the slot therefore (block 50), as well as the stained whole blood sample. The microcontroller 24 senses it, and then actuates a pump that sucks in the blood sample from its vial and adds the correct amount of lysis buffer from a storage reservoir thereof, and then pumps it through a sample loop to allow enough time for incubation within the tube to occur (block 52.) The microcontroller 24 then actuates a pump that pumps the stained and lysed blood sample through the outside microchannels of the passageway of the chip 20, and also pumps a clean buffer solution from its respective reservoir and delivers it to the center microchannel of the passageway of the chip (blocks 54,56). The chip will then passively and centrally focus the stained white cells into the clean buffer stream (block 58), whereupon the washed lysed cells are exported to a vial for subsequent input into to a flow cytometer (block 60), while the waste is delivered to a waste-storage receptacle or reservoir (block 62).

In Fig. 6, the flow chart for the operation of the microcontroller of the device of Fig. 3 is shown. Initially, one first manually inserts the wash-station chip 20 into the slot therefore (block 66), as well as the stained, lysed blood sample. The microcontroller 24 senses it, and then actuates pumps that suck in the blood sample from its vial and then pumps the stained and lysed blood sample through the outside microchannels of the passageway of the chip 20, and also pumps a clean buffer solution from its respective reservoir and delivers it to the center microchannel of the passageway of the chip (blocks 68, 70). The chip will then passively focus the stained white cells into the clean buffer stream (block 72), whereupon the washed lysed cells are exported to a vial for subsequent input into to a flow cytometer (block 74), while the waste is delivered to a waste-storage receptacle or reservoir (block 76) Referring to Fig. 7, there is shown the passageway contained interiorly of, and formed in, the chip or cartridge 20. The chip 20 has an outer, rectilinear- shaped housing, defining therein the passageway 80 having a linear inlet-passageway 80' into which the stained and lysed blood sample and the clean buffer stream are pumped, with the lysed particle stream flowing along the outside microchannels at the wall surfaces of the inlet-passageway, while the clean-buffer solution stream flows along the center microchannel thereof. The heart of the passageway is a reversing spiral, having N amount of spiral arms or turns, Ni amount of inlet or incoming turns, N₂ amount of turns of outlet or outgoing turns, where Ni may be, may not be, equal to N_2.. In the preferred embodiment, three inlet turns or arms 82 and three outlet turns or arms 84 are provided, which terminate into the outlet passageway 86.

The flow in the inlet passageway is as shown in Fig. 8, where the stained, lysed blood stream containing particles flows along the outside walls surfaces or outside microchannels, owing to the experiencing of Dean turbulent or vortex flow, while the clean buffer fluid stream flows along the center of the passageway or the center channel because of laminar flow, which laminar flow occurs since the channels are small and the Reynolds number is low. The particles are moved across the laminar flow by inertial lift forces. At the outlet-passageway, after inertial microfluidic focusing and separation have been achieved in the spiral passageway, the washed cell stream containing only stained white cells, or particles, with clean buffer flow along the center channel of the exit or outlet passageway 86, where the red-blood remnants and reagents flow along the wall surfaces in the outside microchannels for subsequent disposal. The chip 20 saves considerable time, in that a conventional centrifuge that is presently used, typically takes 10 minutes or more, whereas the flow through chip 20 takes on the order of 1 minute to do a sample.

As one example of use of the apparatus 10, one common assay is CD4+ for HIV progression. Hithertofore, sample preparation and assaying has been done manually by a technician, in which he starts with a whole blood sample. The technician first pipettes 50μ1 of blood into a tube, and then pipettes 1ml of lysis buffer into the tube to eliminate the red blood cells and save the white cells. The technician adds cell stains to label the white blood cell types for CD4+, and then will spin the tube in a centrifuge to pull the cells from the lysis buffer and stain. The technician then re-suspends the cells in clean buffer before giving the tube to another technician to run through a cytometer to obtain cell-type counts. The apparatus 10 replaces part or all of these steps. Thus, as described above, for the full-operation embodiment of the apparatus 10 of Figs. 1 and 4, a blood sample will be pumped into the device and an assay chosen from the stored, chilled stains. The device will mix correct amounts of reagent, lysis buffer, and blood sample and incubate for the correct amount of time. This sample, via control of the microcontroller 24, will then be pumped through the washing cartridge, where clean buffer will flow through the center microchannel. The stained CD4+ cells will migrate passively from the outside microchannels into the clean buffer solution, and are then exported into an awaiting sample vial for use in a flow cytometer.

In this specific assay, the cartridge 20 has a passageway that has a cross- sectional shape that is rectilinear, with the following approximate dimensions: A height of between 50μιη and ΙΟΟμιη, a width of between ΙΟΟμιη and 200 μιη, and an overall length of the spiral- section 82, 84 of the passageway being approximately in the range of 1.5 cm. and 2.5 cm,., and preferably approximately 2.0 cm. The length of the linear inlet section 80' is approximately 1.5 cm., while the length of the linear outlet 86 is also approximately 1.5 cm. These characteristics of the passageway 80 will also separate and focus other assays besides that for the CD4+, this being given only by way of example, it being understood that different dimensions and different configurations of the passageway 80 are possible and necessitated by the environment and art in which it is intended to be used, and the stained or labeled particles to be separated out and washed. For this configuration, the Reynolds numbers is in the range of between 0.1 and 20, while the Dean Number is between 1 and 10. A simple, straight or linear passageway may also be used effectively, although the above-described multi-arm, reversing spiral passageway is the preferred, as discussed hereinbelow.

Two configurations have been evaluated for the passageway 80 for use in the apparatus 10: One, a high-aspect ratio rectangular linear channel; and the other, a low-aspect ratio reversing spiral channel 80, as discussed hereinabove, that passively focus particles and cells to precise positions within the center microchannel.

The two basic microfluidic channel configurations for passive, inertial focusing of particles for cell washing are created using standard soft lithography techniques in PDMS. In the first type of configuration based on a simple rectangular channels, the design is a single, straight-flow channel approximately 14μιη wide, 30μιη tall, and 4.5 cm. in length, and is used for focusing in the simplest way. Also, the passageway may be also a 16 μιη wide and 37 μιη high- aspect ratio, tall channel on a single device. The final straight, rectangular configuration includes larger channels optimal for 10 μιη particles, with a channel width of 44 μιη and height of 55 μιη. This design provides similar results as the other channel. The second focusing channel configuration, the reversing spiral pattern 80 with 6 turns, descrived hereinabove, in which the particles flow in a spiral toward the center, and then back out from the center, are intended for two particle regimes. The first spiral channel, which focused 10 μιη particles well, is 40 μιη deep and 100 μιη wide with an outside spiral diameter of 1.7 cm. The second spiral channel, which focuses smaller particles, was 20 μιη deep and 50 μιη wide with an outside spiral diameter of 1 cm. Particle samples are connected using 1/16" OD x 0.030" ID PEEK tubing (IDEX, Corp., IL). Volumetric flow rates range from 40 μΐ/min to 500 μΐ/min depending on the particle size and the channel dimensions.

In one assay for standard CD4+ panleukogating, the following preparation is to be used for performing standard protocols in the apparatus 10: RBC Lysis buffer (10X), 420301, (Biolegend, San Diego, CA) along with the Cell Staining buffer, 420201 , (Biolegend, San Diego, CA). The anti-human CD4 antibody conjugated to Phycoerythrin (PE) and the anti-human CD45 antibody would be conjugated to PE.Cy5 (Biolegend , San Diego, CA). 100 μΐ, of IMMUNO-TROL, (Beckman Coulter, Brea, CA) positive process control cells would then be incubated with 10 μL of each of the antibodies listed above for 20 minutes in the dark. The stained whole blood is then lysed for 10 minutes in 1.5 mL of lysis buffer. The lysed stained cells are is then separated, focused, and washed at the wash station 42 via the microchannels of the passageway 80 of the cartridge 20, in the manner described hereinaabove.

Regarding the low-aspect spiral channels, using a symmetric spiral in and spiral out pattern, as described above, such a reversing spiral results in improved focusing accuracy due to mixing action of Dean flow allowing particles to access dynamic equilibrium positions more quickly. Additionally, for low-aspect ratio channels (much wider than tall), the secondary Dean flow results in a shift of the two equilibrium positions of focused particles towards the inner bend of the curving channel and towards the centerline in the vertical direction.

Two other curved configurations may also be employed. The first uses a combined a series of sigmoidal curves with a second stage, high-aspect ratio channel to create a focusing system. The other is a single direction spiral for the positioning of a single type of cultured cells and a single size of microspheres. The use of curved low-aspect channels may also be advantageous because of their easier manufacturability for mass-produced thermoplastic devices.

With regard to the reversing-spiral passageway 80, one that is 100 μιη wide, 40 μιη tall, with 6-turn reversing spiral, focused 10 μιη particles well, but does not focus smaller particles as well when flowing at 150 μΐ/min. Nonetheless, this design does result in central positioning of particles of a variety of sizes. For 4 μιη and 6 μιη particles, only slight focusing occurs. However, when 10 μιη particles are passed through the channel, they are focused tightly. To improve the focusing of particles smaller than 10 μιη in diameter, decreasing of the channel and spiral dimensions by half is employed, creating 50 μιη wide by 20 μιη tall channels. Using this smaller channel size, 4 μιη and 6 μιη particles are successfully focused. Thus, a smaller channel or focusing for a longer channel length is implemented for higher precision focusing of 4 μιη particles. However, with regards to the 6 μιη particles, excellent focusing is achieved.

Using the example given hereinabove, the passageway 80 with the reverse spiral channels is used for CD4+ cell assays. For the reversing spiral channels, a flow rate of 50 μΐ/min is used through the spiral device. The channel dimensions of this device are 50 μιη wide by 20μιη high, which can effectively position cells used (lymphocytes ~8 μιη, monocytes -15 - 20 μιη, and granulocytes -9 - 15 μιη) to the centerline for inclusion in the clean buffer wash stream.

It is also within the scope and purview of the invention to provide a device 10 that utilizes a plurality or series of chips or cartridges 20 by which multiple samples may be processed simultaneously.

In addition, the method and apparatus of the invention may also used for bead-based assays used in the drug industry for the discovery of new drugs. Conventional bead based assays capture cells or proteins/other biological chemicals from solutions. Conventional magnetic bead based assays have a sample full of cells, have biotinalated beads and a strepatavitin antibody for a specific cell type. The beads are then mixed into the sample. The beads stick to the cells of interest and then are pulled out with a magnet. Centrifugal polystyrene or ceramic beads methods spin the solution down using a centrifugal device and pull off the supernatant, leaving only the beads with the protein of interest attached.

In bead-based assays, the same procedure is generally followed as described hereinabove with regard to the stained and lysed sample, except that instead of cells or particles being separated and focused to a center buffer stream in the center microchannel, beads with antibodies are acted upon, for subsequent delivery to a flow cytometer. Hithertofore, separation has been done by centrifuging to a wash. For example, the apparatus of the present invention, or at least the chip 20, may replace part or all of Invitrogen's Multiplex Bead Immunoassay Kits that are developed to maximize flexibility in experimental design, permitting the measurement of one or multiple proteins in panels designed by a researcher, and Invitrogen's Human Cytokine Magnetic 10-Plex Panel that contains all the reagents that are intended for use with the LuminexR 100™, 200™ or FlexMAP 3DR dual laser detection system.

The method of the invention may be used in any environment where a center buffer-wash stream is to be used to wash or clean biological particles or cells. Examples of this are diluted blood sample streams where inertial focusing pulls or draws the cells from the outer microchannels to the center buffer- wash stream, or for isolating rare cells, such as circulating tumor cells, so as to accumulate a lysed sample and running it through the chip of the invention, whereby the cells desired to be cleaned are inertially focused into the center clean-buffer stream and thus away from background debris or solutions.

While a specific embodiment of the invention has been shown and described, it is to be understood that numerous changes and modifications may be made therein without departing from the scope and spirit of the invention.

Claims

IN THE CLAIMS:

CLAIM 1. A method of washing a stained, lysed blood sample, comprising:

(a) directing the stained, lysed blood sample to the outside microchannels of a microfluidic passageway for developing a flow stream thereat;

(b) directing a clean buffer solution to the center microchannel of the microfluidic passageway for developing a flow stream thereat;

(c) passively separating the stained white blood cells from the lysed blood sample of said step (a) in said microfluidic passageway, and focusing the stained white blood cells to the center microchannel for mixing with said clean buffer solution stream in said center microchannel;

(d) outputting the resultant mixture of clean buffer and stained white blood cellsr use with a flow cytometer;

(e) outputting the waste stream in said outside microchannels for subsequent disposal;

(f) said step (c) comprising inertial microfluidic separation in said microfluidic passageway.

CLAIM 2 . The method of washing a stained, lysed blood sample according to claim 1, wherein, said steps (a) and (b) comprise delivering the said respective streams to a reverse spiral microfluidic passageway having an inlet, an outlet, and a main reverse spiral section.

CLAIM 3 . The method of washing a stained, lysed blood sample according to claim 1, wherein, said steps (a) and (b) comprise delivering the said respective streams to a reverse spiral microfluidic passageway having an inlet, an outlet, and a main reverse spiral section; said main reverse spiral section having N amount of spiral arms, where N comprises an Ni amount of inlet turns, and N₂ amount of outlet turns, where Ni may be, or may not be, equal to N_2.

CLAIM 4. A method of washing a sample containing biological cells and particles, for subsequent delivery to a flow cytometer, comprising:

(a) directing the sample to the outside microchannels of a microfluidic passageway for developing a flow stream thereat;

(b) directing a clean buffer solution to the center microchannel of the microfluidic passageway for developing a flow stream thereat; (c) passively separating the biological cells or particles from the sample of said step (a) in said microfluidic passageway, and focusing the biological cells or particles to the center microchannel for mixing with said clean buffer solution stream in said center microchannel;

(d) outputting the resultant mixture of clean buffer and biological cells or particles for use with a flow cytometer;

CLAIM 5 . The method of washing a stained, lysed blood sample according to claim 4, wherein, said steps (a) and (b) comprise delivering the said respective streams to a reverse spiral microfluidic passageway having an inlet, an outlet, and a main reverse spiral section.

CLAIM 6 . The method of washing a stained, lysed blood sample according to claim 4, wherein, said steps (a) and (b) comprise delivering the said respective streams to a reverse spiral microfluidic passageway having an inlet, an outlet, and a main reverse spiral section; said main reverse spiral section having N amount of spiral arms, where N comprises an Ni amount of inlet turns, and N₂ amount of outlet turns, where Ni may be, or may not be, equal to N_2.

CLAIM 7. An apparatus for washing a sample containing biological cells or particles, such as a stained, lysed blood sample, beads containing biological cells and particles, and the like, for subsequent delivery to a flow cytometer, comprising: a main housing;

at least one wash-chip comprising a main frame defining an interior portion, and a microfluidic passageway formed in said interior portion, said microfluidic passageway comprising outside microchannels and a center microchannel;

said main housing having at least one chip-input station for removably and interchangeably receiving therein said at least one wash-chip;

a first input station for receiving a sample containing biological cells, or particles, to be separated out from said sample, and being in fluid communication with said at least one chip-input station; a second input station for receiving a clean-wash buffer solution for the separated biological cells or particles, and being in fluid communication with said at least one chip-input station ;

said first input station comprising a pump for pumping the sample containing biological cells or particles to said outside microchannels of said microfluidic passageway;

said second input station comprising a pump for pumping the clean-wash buffer solution to said center microchannel of said microfluidic passageway;

said microfluidic passageway performing inertial microfluidic separation and focusing of the cells or particles in the sample to said center microchannel for mixing with said clean-wash buffer solution;

said microfluidic passageway comprising an outlet for said mixture of cells or particles with clean- wash buffer solution for subsequent analysis thereof.

CLAIM 8. The apparatus for washing a sample containing biological cells or particles according to claim 7, wherein said microfluidic passageway comprises a reverse spiral microfluidic passageway having an inlet, an outlet, and a main reverse spiral section.

CLAIM 9. The apparatus for washing a sample containing biological cells or particles according to claim 8, wherein said reverse spiral microfluidic passageway comprises an inlet, an outlet, and a main reverse spiral section; said main reverse spiral section having N amount of spiral arms, where N comprises an Ni amount of inlet turns, and N₂ amount of outlet turns, where Ni may be, or may not be, equal to N₂.

CLAIM 10. The apparatus for washing a sample containing biological cells or particles according to claim 8, wherein said microfluidic passageway comprises a low-aspect ratio, rectilinear- shaped cross-sectional microfluidic passageway.

CLAIM 11. The apparatus for washing a sample containing biological cells or particles according to claim 8, wherein said microfluidic passageway comprises a high-aspect ratio, rectilinear-shaped cross-sectional microfluidic passageway.

CLAIM 12. The apparatus for washing a sample containing biological cells or particles according to claim 8 , wherein said microfluidic passageway comprises a linear microfluidic passageway.

CLAIM 13. The apparatus for washing a sample containing biological cells or particles according to claim 8, wherein said microfluidic passageway has a height of between 50μιη and ΙΟΟμιη, a width of between ΙΟΟμιη and 200 μιη, and an overall length of said main reverse spiral section being approximately in the range of 1.5 cm. and 2.5 cm.

CLAIM 14. The apparatus for washing a sample containing biological cells or particles according to claim 13, wherein the flow in said microfluidic passageway has a Reynolds numbers in the range of between 0.1 and 20, and a Dean Number between 1 and 10.

CLAIM 15. The apparatus for washing a sample containing biological cells or particles according to claim 7, comprising a plurality of said wash-chips; and said main housing comprising a plurality of chip-input stations, whereby a plurality of samples may be processed simultaneously.

CLAIM 16. A method of washing a sample containing biological cells or particles, such as a stained, lysed blood sample, beads containing biological cells and particles, and the like, for subsequent delivery to a flow cytometer, comprising:

(a) delivering the sample to at least one first input station;

(b) delivering the sample to least one wash-chip comprising a main frame defining an interior portion, and a microfluidic passageway formed in said interior portion, said microfluidic passageway defining outside microchannels and a center microchannel;

(c) said step (b) comprising delivering the sample to the outside microchannels of said passageway;

(d) delivering a clean-buffer wash solution to a second input station for receiving the clean-buffer wash solution thereat;

(e) delivering the clean-buffer wash solution to the at least one wash-chip of said step (b);

(f) said step (e) comprising delivering the clean-buffer wash solution to the center microchannel of said microfluidic passageway;

(g) said steps (b), (c), (e) and (f) passively separating said biological cells or particles from said sample and focusing them into said center microchannel of said microfluidic passageway for mixing with said clean-buffed wash solution.

CLAIM 17. The method of washing a sample containing biological cells or particles according to claim 16, further comprising outputting the mixture of clean-buffer wash solution and focused cells or particles after said steps (b) through (g) for analysis thereof; and outputting the waste stream in said outside microchannels after said biological cells or particles have been separated from said sample, for subsequent disposal.

CLAIM 18. The method of washing a sample containing biological cells or particles according to claim 17, wherein said step of outputting the mixture of clean-buffer wash solution and focused cells or particles after said steps (b) through (g) for analysis comprising delivering the mixture of clean-buffer wash solution and focused cells or particles to a flow cytometer.

CLAIM 19. A method of washing a biological sample from a biological mixture, such as a stained, lysed blood sample, a bead-based assay with captured cells, proteins or other biological chemicals from a solution, comprising:

(a) directing a biological mixture comprising a biological sample to be separated out to the outside microchannels of a microfluidic passageway for developing a flow stream thereat;

(c) passively separating the biological sample from the biological mixture of said step (a) in said microfluidic passageway, and focusing them to the center microchannel for mixing with said clean buffer solution stream in said center microchannel;

(d) outputting the resultant mixture of clean buffer and biological sample for subsequent use;

(e) outputting the resultant waste stream in said outside microchannels for subsequent disposal;

CLAIM 20. The method of washing a biological sample from a biological mixture according to claim 19, further comprising:

(g) wherein said step (d) comprises outputting the resultant mixture of clean buffer and biological sample to a flow cytometer;