WO2010080978A2

WO2010080978A2 - Pre-depletion of leukocytes in whole blood samples prior to the capture of whole blood sample components

Info

Publication number: WO2010080978A2
Application number: PCT/US2010/020470
Authority: WO
Inventors: Sunitha Nagrath; Mehmet Toner
Original assignee: The General Hospital Corporation
Priority date: 2009-01-08
Filing date: 2010-01-08
Publication date: 2010-07-15
Also published as: WO2010080978A3

Abstract

Methods, apparatuses, and microfluidic systems for pre-depletion of leukocytes in whole blood samples prior to the capture of whole blood sample components is described. A microfluidic device is used to pre-deplete the leukocytes in a whole blood sample. To do so, parallel micro-channels in the microfluidic device are treated with binding moieties to bind leukocytes but not target cells. When the whole blood sample is flowed through the microfluidic device, it is depleted of the leukocytes to form a depleted sample. The depleted sample is then flowed through a second micro-channel treated to bind target cells.

Description

PRE-DEPLETION OF LEUKOCYTES IN WHOLE BLOOD SAMPLES PRIOR TO THE CAPTURE OF WHOLE BLOOD SAMPLE COMPONENTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Patent Application Serial No. 61/143,316, filed on January 8, 2009, the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] This disclosure relates the selective capture of components, such as rare living cells, from biological samples, such as blood.

BACKGROUND

[0003] The isolation of specific cell populations from complex mixtures such as whole blood has significant utility in both clinical practice and basic medical research. A variety of approaches may be used to separate cells from a heterogeneous sample. For example, some techniques can use functionalized materials to capture cells by binding cell surface markers that are particular to the target cell population. The functionalized materials can include surface-bound capture moieties such as antibodies or other specific binding molecules, such as aptamers or selectins.

[0004] Viable tumor-derived epithelial cells (for example, circulating tumor cells or CTCs) have been identified in peripheral blood from cancer patients and are likely the origin of intractable metastatic disease. CTCs represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization, and monitoring of non-hemato logic cancers. The ability to identify, isolate, propagate, and molecularly characterize CTC subpopulations could further the discovery of cancer stem cell biomarkers and expand the understanding of the biology of metastasis. Current strategies for isolating CTCs are limited to complex analytic approaches that generate very low yield and purity. CTCs are considered to be rare, making up as few as about 1 CTC per 10⁹ hematologic cells in the blood of patients with metastatic cancer. The isolation of CTCs from blood samples presents a tremendous technical challenge.

[0005] Micro fluidic lab-on-a-chip devices provide unique opportunities for cell sorting and rare cell detection. Such devices have been successfully used for microfluidic flow cytometry, continuous size-based separation, and chromatographic separation. For example, a microfluidic affinity-based chip that is configured to isolate CTCs from the whole blood of cancer patients is described, for example, in Nagrath et al., "Isolation of rare circulating tumour cells in cancer patients by microchip technology," Nature 450 (2007), pp. 1235-1239. CTCs may disseminate from the tumor and are observed to be present in numbers that tend to correlate with patients' clinical courses. CTCs may also be involved in metastasis. Accordingly, such microfluidic chip technology may be used in diagnostic and prognostic devices for oncological applications.

SUMMARY

[0006] This disclosure provides methods and microfluidic devices for pre-depletion of leukocytes in whole blood samples prior to capture of whole blood sample components.

[0007] In general, one innovative aspect of the subject matter described in this disclosure can be implemented as a method for capturing target cells from whole blood. A first set of multiple micro-channels are formed on a microfluidic device. The set of micro-channels are parallel to each other and have a common inlet and a common outlet. Each micro-channel in the first set of micro-channels is configured to bind leukocytes. The first set of micro-channels is coupled with a second micro-channel configured to bind target cells. A sample of whole blood including leukocytes and the target cells is flowed through the first set of micro-channels. The first set of micro-channels bind the leukocytes such that the sample is depleted of leukocytes to form a depleted sample. The depleted sample is flowed through the common outlet and into the second micro-channel. The second micro-channel binds the target cells.

[0008] This, and other aspects, can include one or more of the following features. Configuring each micro-channel in the first set of micro-channels to bind leukocytes can include treating each micro-channel with CD2 configured to bind lymphocytes. Flowing the sample of whole blood including leukocytes and the target cells through the first set of micro-channels can further include flowing the sample of whole blood at a shear stress substantially equal to 1.75 dynes/cm² to bind the lymphocytes. Configuring each micro-channel in the first set of micro-channels to bind leukocytes can include treating each micro-channel with CD66 configured to bind neutrophils. Flowing the sample of whole blood including leukocytes and the target cells through the first set of micro-channels further includes flowing the sample of whole blood at a shear stress substantially equal to 0.45 dynes/cm² to bind the neutrophils. Configuring each micro-channel in the first set of micro-channels to bind leukocytes can include treating each micro-channel with CD45 configured to bind leukocytes. Flowing the sample of whole blood including leukocytes and the target cells through the first set of micro-channels can further include flowing the sample of whole blood at a shear stress substantially equal to 0.82 dynes/cm² to bind the leukocytes. The target cells can include circulating tumor cells. Surfaces of the second micro-channel can be functionalized with EpCAM antibodies using Avidin-Biotin chemistry such that the functionalized surfaces bind the circulating tumor cells. A second set of multiple micro-channels can be formed. Each micro-channel in the second set of multiple micro- channels can be configured to bind to leukocytes. The common outlet of the first set of multiple micro-channels can be coupled to a common inlet of the second set of multiple micro-channels. A common outlet of the second set of multiple micro-channels can be coupled to the second micro-channel. The whole blood sample can be flowed through the first set of multiple micro- channels and the second set of multiple micro-channels to form the depleted sample. The second micro-channel can be formed on a separate microfluidic device. Coupling the first set of multiple micro-channels with the second micro-channel can include providing a fluid connection between the common outlet of the first set of multiple micro-channels to an inlet of the second micro-channel using a capillary tube. Further, IX PBS can be flowed through the first set of multiple micro-channels after flowing the sample of whole blood. The IX PBS can unbind the leukocytes from the first set of multiple micro-channels, thereby preparing the micro-channels for re-use.

[0009] Another innovative aspect of the subject matter described in this disclosure can be implemented as an apparatus for capturing target cells from whole blood. The apparatus includes a microfluidic device including a first set of multiple parallel micro-channels having a common inlet and a common outlet. The first set of multiple micro-channels is treated with a first binding moiety such that the first set of multiple micro-channels bind leukocytes. The apparatus includes a second micro-channel including an inlet and an outlet. The second micro- channel is treated with a second binding moiety such that the second micro-channel binds target cells. The apparatus includes an interface coupling the common outlet of the first set to the inlet of the second micro-channel. A whole blood sample including leukocytes and the target cells that is flowed into the common inlet of the first set flows through the first set of parallel micro- channels, through the common outlet of the first set, and into the inlet of the second micro- channel through the interface such that the whole blood sample flowing through the common outlet of the first plurality is depleted of leukocytes.

[00010] This, and other aspects, can include one or more of the following features. The first set of micro-channels can consist of sixteen micro-channels. Each of the first set of micro- channels can be 50 μm deep, 4 mm wide, and 14 mm long. The first binding moiety can be CD66. The dimensions of each of the first set of micro-channels can be selected to exert a shear stress of 0.45 dynes/cm² on the whole blood sample at a whole blood sample flow rate of 30 μL/min. The first binding moiety can be CD2. The dimensions of each of the first set of micro- channels can be selected to exert a shear stress of 1.75 dynes/cm² on the whole blood sample at a whole blood sample flow rate of 100 μL/min. The first binding moiety can be CD45. The dimensions of each of the first set of micro-channels can be selected to exert a shear stress of 0.82 dynes/cm² on the whole blood sample at a whole blood sample flow rate of 50 μL/min. The second micro-channel can be formed on the microfluidic device. The second micro-channel can be formed on a second microfluidic device that is separate from the microfluidic device. The target cells can be circulating tumor cells. The second microfluidic device can be 25 mm x 66 mm. The second microfluidic device can have an active capture area of 10 mm x 51 mm. The active capture area can include multiple micro-posts. The second binding moiety can include EpCAM antibodies. Each micro-post can be treated with EpCAM antibodies using Avidin- Biotin chemistry such that the micro-posts bind the circulating tumor cells. The capture area can include 78,000 micro-posts. Each micro-post can be 100 μm tall and 100 μm in diameter. The multiple micro-posts can be arranged in an equilateral triangular array with a 50 μm gap between each micro-post. The interface can be a capillary tube. The interface can be detached from the common outlet of the first set and the inlet.

[00011] In any of the foregoing aspects, the binding moieties can be selected from antibodies, antibody fragments, oligo- or poly-peptides, nucleic acids, cellular receptors, ligands, aptamers, MHC-peptide monomers or oligomers, biotin, avidin, oligonucleodites, coordination complexes, synthetic polymers, and carbohydrates. [00012] Also, in any of the foregoing aspects, the sample can be a whole blood sample, the binding moiety can bind to CD45, CD2, CD66, CD14, CD4, CD8, EpCAM, E-Selectin, or P- Selecting. The target cells to be isolated can be selected from neutrophils, monocytes, lymphocytes, CTCs, HIV infected CD8 lymphocytes, circulating endothelial cells, and platelets. In aspects in which the target cells are CTCs, the whole blood sample can be obtained from a patient having or at a risk for cancer.

[00013] Other aspects can include analyzing at least one property (for example, biological property) of the desired cells (for example, MRNA expression, protein expression, DNA quantification, DNA sequence, and chromosomal abnormalities) and/or counting the target cells, for example, to diagnose a disease state such as cancer, for example, when the target cells are CTCs.

[00014] By a "patient" is meant a living multicellular organism. The term "patient" includes humans, mice, dogs, cats, cows, sheep, horses, non-human primates, fish, and the like.

[00015] By "cell surface marker" is meant a molecular bound to a cell that is exposed to the extracellular environment. The cell surface marker can be a protein, lipid, carbohydrate, or some combination of the three. The term "cell surface marker" includes naturally occurring molecules, molecules that are aberrantly present as the result of some disease condition, or a molecule that is attached to the surface of the cell.

[00016] By "lysis" is meant disruption of the cellular membrane. The term "lysis" is meant to include complete disruption of the cellular membrane ("complete lysis"), partial disruption of the cellular membrane ("partial lysis"), and permeabilization of the cellular membrane.

[00017] By "binding moiety" is meant a chemical species to which an analyte binds. A binding moiety can be a compound coupled to a surface or the material making up the surface. Exemplary binding moieties include antibodies, antibody fragments (for example, Fc fragments), oligo- or poly-peptides, nucleic acids, cellular receptors, ligands, aptamers, MHC-peptide monomers or oligomers, biotin, avidin, oligonucleotides, coordination complexes, synthetic polymers, and carbohydrates. [00018] By "permeabilization" is meant the disruption of the cellular membrane such that certain intracellular components are able to escape the cell, while other components remain inside the cell.

[00019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art that this disclosure describes.

[00020] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the techniques described in the present disclosure, useful methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflicting subject matter, the present disclosure, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[00021] Particular embodiments of the subject matter described in this disclosure can be implemented to realize one or more of the following potential benefits. The methods and apparatus described here can be employed to deplete leukocytes in whole blood samples that include leukocytes and target cells, for example, CTCs, prior to the capture of the target CTCs. The micro fluidic techniques can be used to increase the average capture purity of target cells. The average capture purity is defined as percentage ratio of number of target cells to non- specifically bound leukocytes. Not only can the average capture purity of CTCs be increased but also the variation across samples can be decreased. Decreasing the range of capture purity across multiple samples can enable using direct sequencing for the majority of the sample, rather than using allele-specific polymerase chain amplification for epidermal growth factor receptor (EGFR) mutational analysis. The microfluidic apparatus described here can capture and isolate very pure subpopulations of white blood cells directly from whole blood. By pre-depleting the contaminant leukocytes prior to capturing CTCs, the average capture purity can be increased by orders of magnitude. For example, the post-depletion EpCAM capture of CTCs increases in proportion to the depletion of leukocytes using CD45. This can enable the genotyping and phenotyping of CTCs which, in turn, can provide detailed insight into the metastatic process and permit direct exploration of targeted treatment strategies. Furthermore, despite the rarity of the CTC in the sample and/or reduced levels of CTC expression of the antigen corresponding to the surface-bound antibody, the capture of CTCs can be improved.

[00022] These and other objects, features and advantages of the disclosure will become apparent upon reading the following detailed description of embodiments, when taken in conjunction with the appended claims. The details of one or more embodiments are set forth in the accompanying drawings and the description below.

BRIEF DESCRIPTION OF THE DRAWINGS

[00023] Figure 1 is a schematic diagram showing a system to pre-deplete leukocytes in whole blood samples prior to capturing target cells.

[00024] Figure 2 is a schematic diagram showing a microfluidic device including multiple micro-channels to bind leukocytes in whole blood samples.

[00025] Figure 3 is a schematic diagram showing a micro-channel including multiple micro-posts to bind CTCs.

[00026] Figure 4 is a schematic diagram showing a microfluidic device to deplete leukocytes coupled with a micro-channel to bind CTCs coupled with a capillary tube interface.

[00027] Figure 5 is a schematic diagram showing multiple micro-channels to deplete leukocytes and a micro-channel to bind CTCs formed on the same microfluidic device.

[00028] Figure 6 is a table of cell populations, capture antibodies, and shear stresses to bind a cell population to a corresponding capture antibody.

[00029] Figures 7A and 7B show an image of a microfluidic device to capture leukocytes and capture efficiencies of the device at different volumetric flow rates.

[00030] Figure 8 shows a chart of non-specific binding of target cells in the micro- channels at varying volumetric flow rates.

[00031] Figure 9 is a chart showing comparing non-specific binding per unit volume of whole blood sample in the presence of and absent pre-depletion. [00032] While methods and apparatuses will now be described in detail with reference to the figures, they are done so in connection with the illustrative embodiments and are not limited by the particular embodiments illustrated in the figures.

DETAILED DESCRIPTION

[00033] The present disclosure describes methods and devices for the isolation of analytes

(for example, target cells). A whole blood sample including target cells is introduced into a geometry of micro-channels containing moieties that bind leukocytes, thereby depleting the whole blood sample of leukocytes. A shear stress is applied that is sufficiently low to allow the micro-channels to bind and retain the leukocytes. The depleted sample that includes the target cells is subsequently flowed through another micro-channel containing moieties such that the micro-channel binds the target cells. Once bound, the target cells can be analyzed (for example, counted).

[00034] An example of a target cell is a CTC of epithelial origin from peripheral blood.

Other rare cells include organisms potentially found in peripheral blood (for example, bacteria, viruses, protists, or fungi), other nonhemopoietic cells not normally found in blood (for example, endothelial cells or fetal cells), and even cells of hemopoietic origin (for example, platelets, sickle cell red blood cells, and subpopulations of leukocytes). The binding agent or agents employed will depend on the type of cell or cells being targeted. In the description that follows, CTCs are the target cells that are captured subsequent to pre-depletion of leukocytes from whole blood samples. It will be appreciated that other target cells can be captured using appropriate binding moieties.

[00035] Exemplary types of binding moieties include antibodies, antibody fragments (for example, Fc fragments), oligo- or polypeptides, nucleic acids, cellular receptors, ligands, aptamers, MHC-peptide monomers or oligomers, biotin, avidin, oligonucleotides, coordination complexes, synthetic polymers, and carbohydrates. Binding moieties may be attached to chambers, for example, inner surfaces of micro-channels formed in micro fluidic devices, using methods known in the art. The method employed will depend on the binding moiety and the material used to construct the device. Examples of attachment methods include non-specific adsorption to the surface, either of the binding moiety or a compound to which the binding moiety is attached or chemical binding, for example, through self assembled monolayers or silane chemistry. A preferred exemplary binding agent is anti-EpCAM antibody, which is specific for epithelial cells. As described, circulating epithelial cells may provide clinical and diagnostic information relevant to tumors, even those considered clinically localized.

[00036] For pre-depleting the leukocytes from whole blood, various binding moieties such as antibodies or combinations of antibodies such as, for example, CD2 (lymphocytes), CD66 (neutrophils), CD45 (leukocytes). As explained later, the inner surfaces of the micro-channels of the microfluidic device are treated with the binding moieties. Subsequently, whole blood samples that include the leukocytes are flowed through the micro-channels at particular volumetric flow rates such that the shear stresses exerted on the leukocytes are sufficiently low to bind the leukocytes (or specific components of the leukocytes) to the treated surfaces of the micro-channels. In the techniques described below, the binding moiety used to capture the leukocytes consists of CD45. Accordingly, the micro-channels are treated with CD45 to pre- deplete the leukocytes from the whole blood samples. It will be appreciated that other binding moieties can be used to capture the leukocytes.

[00037] Figure 1 is a schematic diagram showing a system 100 to pre-deplete leukocytes in whole blood samples prior to capturing target cells. The system 100 includes a microfluidic device 105 on which multiple micro-channels 115 are formed. The multiple micro-channels 115 include a common inlet 120 and a common outlet 125. As described later, the micro-channels 115 are treated with a binding moiety to bind leukocytes in a whole blood sample 105 that includes leukocytes and target cells. The binding moieties are selected such that the target cells in the whole blood sample 105 do not bind to the micro-channels 115. In some implementations, the whole blood sample 105 is flowed through the common inlet 120 and the micro-channels 115 at volumetric flow rates selected to exert particular shear stresses on the whole blood sample 105. The particular shear stresses increase the likelihood of leukocytes coming into contact with and binding to the inner surfaces of the micro-channels 115. Consequently, the whole blood sample 105 that flows past the common outlet 125 is depleted of leukocytes. The binding force between the antigen and antibody coated surface depends on the bio-specific interaction between the antigen and antibody. The ability of adhesion molecules to support cell adhesion under flow, and the dynamic mode of adhesion, is determined by the physiochemical parameters of the system, including binding kinetics, their response to stress, and their site densities on the cell and surface. Hence, using shear force, the binding of cells to specific antigens can be regulated.

[00038] The system 100 includes a second micro-channel 130 that is configured to bind the target cells. As described later, the depleted whole blood sample 105 is flowed from the common outlet 125 into the second micro-channel 130. The target cells bind to the second micro-channel 130. The design of the second micro-channel 130 and the flow rates at which the depleted whole blood sample 105 is flowed through the second micro-channel 130 increase an average purity ratio (percentage ratio of number of target cells to non-specifically binding leukocytes) of the captured CTCs. Pre-depletion of the leukocytes in the whole blood sample 105 prior to flowing the sample 105 through the second micro-channel 130 decreases the sample- to-sample variability of the purity ratio. In some implementations, additional micro fluidic devices, such as micro fluidic device 135, in which micro-channels are formed, can be used to additionally pre-deplete leukocytes prior to target cell capture using the second micro-channel 130.

[00039] Figure 2 is a schematic diagram showing a micro fluidic device 110 including multiple micro-channels to bind leukocytes in whole blood samples. In some implementations, the micro-channels 115 are parallel to each other. Each micro-channel 115 is 14 mm long, 4 mm wide, and 50 μm deep. In some implementations, the microfluidic device 110 is fabricated from polydimethylsiloxane (PDMS) using standard molding techniques, and consists of sixteen individual micro-channels 115 connected through a fluidic circuit as shown in figure 2. The well-defined flow within each micro-channel 115 can allow precise control of shear forces experienced by cells at the surface of the microfluidic device 110. Combined with the specificity of monoclonal antibodies for cell surface antigens, the control provides conditions under which the capture of specific cells from blood can be enhanced. In some implementations, the channel dimensions can be fixed and the flow rates can be varied to obtain various shear stresses to determine the flow condition under which capture efficiency is increased. Since, as long as the shear stress is same, the device could of any dimension, it will be appreciated that any microfluidic device can be used for pre-depletion. In some implementations, regardless of the length and width of each micr-channel, the depth of the micro-channel is 50μm. [00040] In some implementations, the whole blood sample 105 including the leukocytes and the target cells, for example, CTCs, is flowed through the common inlet 120, through each micro-channel 115, and through the common outlet 125 using a syringe pump, for example. Depending upon the cell population to be depleted, the volumetric flow rates of the whole blood sample 105 can be controlled to exert particular shear stresses on the whole blood sample 105 (figure 6). For example, when the micro-channels 115 are treated with the binding moiety, CD45, and the volumetric flow rate of the whole blood sample 105 is 50 μL/min, then a shear stress of 0.82 dynes/cm² is exerted on the sample 105 thereby increasing the leukocytes bound to the micro-channels 115. The sample 105 that flows out of the common outlet 125 is depleted of the leukocytes.

[00041] In some implementations, the depleted sample is flowed through the second micro-channel 130 to capture the CTCs. In alternative implementations, the depleted sample can be flowed through another micro fluidic device 135 having micro-channels that can be treated to bind cells other than the CTCs in the depleted sample 105. In some scenarios, the micro- channels of the microfluidic device 135 can be treated to bind the same cells that the micro- channels 115 are treated to bind. Doing so can further deplete the whole blood sample 105 prior to CTC capture. In alternative scenarios, the micro-channels of the microfluidic device 135 can be treated to bind other cells in the whole blood sample 105 that can negatively affect the purity ratio. For example, the micro-channels of the microfluidic device 135 can be treated with the binding moiety, CD2, to bind lymphocytes.

[00042] In this manner, multiple microfluidic devices can be connected in series to pre- deplete the whole blood sample 105 of non-specific binding cells other than the target cells. Typically, the binding moieties are disposed on the walls of the micro-channels, although additional structures, for example, micro-posts, can be included in the channel to increase the surface area. The sample 105 containing non-specific binding cell, for example, neutrophils, lymphocytes, and the like, can be applied at a shear stress preferably low enough to allow binding of the cell. Each microfluidic device in series can remove or decrease one or more types of cells, which are not the target cells. The shear stress applied to each of the chambers can be different (achieved for example by varying the cross sectional area of the chambers) or the shear stress can be the same. Also, each chamber can contain binding moieties that bind to different cell surface markers or the same cell surface markers. When the same binding moiety in employed in different microfluidic devices, the methods may be used to isolate, in series, nonspecific binding cells that have progressively lower amounts of substance to which the binding moiety binds.

[00043] Figure 3 is a schematic diagram showing a micro-channel 130 including multiple micro-posts to bind CTCs. The micro-channel 130 can be formed in a microfluidic device 305, for example, by silicon etching. In some implementations, the dimensions of the device 305 are 25 mm x 66 mm, with an active capture area of 19mm x 5 lmm. The active capture area can include micro-posts 310, for example, arranged in an equilateral triangular array. For example, the array can include 78,000 micro-posts 310 of 100 μm height and 100 μm diameter. Each micro-post 310 can be spaced apart by a gap of approximately 50μm. It will be appreciated that the dimensions of the device 305, the number and dimensions of the micro-posts 310, and the gap between micro-posts 310 are exemplary and variable.

[00044] Figure 4 is a schematic diagram showing a microfluidic device 110 to deplete leukocytes coupled with a micro-channel 130 to bind CTCs coupled with a capillary tube interface 405. In some implementations, the common outlet of the microfluidic device 110 can be coupled to an inlet of a capillary tube to arrange a fluid connection that forms an interface between the two. Similarly, an outlet of the capillary tube can be coupled to an inlet of the second micro-channel 130. The whole blood sample 105 from the leukocytes have been depleted is flowed through the micro-channels 115 of the microfluidic device 110, through the capillary tube interface 405, and into the second micro-channel 130.

[00045] Figure 5 is a schematic diagram showing multiple micro-channels 115 to deplete leukocytes and a micro-channel 130 to bind CTCs formed on the same microfluidic device. In some implementations, the micro-channels 115 and the micro-channel 130 can be formed on the same substrate and can be fluidically connected via another micro-channel formed on the substrate. Alternatively, the micro-channels 115 and the micro-channel 130 can be fluidically connected using capillary tubes.

[00046] An exemplary protocol for a method of pre-depleting leukocytes from whole blood samples by binding the leukocytes to micro-channels 115 treated with CD45 is described herein. The following materials are used to perform the method:

- outlet/waste tubing: 6" tygon tubing with plugs inserted to one end;

- inlet tubing: 8" of tugon tubing connect with needle tip at both ends, attached to 1 "teflon tubing or 9" tygon tubing, needle tip at one end;

- connection tubing from depletion to EpCAM device: 3" tygon tubing with needle tip both end, connected to 3" of teflon tubing;

- connection tubing from depletion to depletion (in tandom/series): 6" tygon tubing with needle tip inserted at both ends;

- waste tubes petri dish ImI syringes 22g needle tips; and

25G needle tips kirn wipes 20ug/ml CD45 PBS (w/o Ca+ and Mg+) pre-warmed 1%BSA, IxPBS, 4%PFA or BD cytofϊx/cytoperm.

[00047] The micro-channels 115 are treated with CD45 to bind the leukocytes by functionalizing the inner surfaces of the micro-channels 115. The micro fluidic device 110, which is stored in Avidin in a cold room, is brought to room temperature (15 minutes). The micro-channels 115 are flushed with 1 mL of IX PBS (1 mL syringe with 22G needle tip) from any inlet to wash out the Avidin. The micro-channels 115 are then flushed with 100-200 μL of 100-200 μg/mL of CD45 (1 mL syringe with 22G needle tip) into the common inlet 125 and incubated for 30 minutes. These steps are repeated through the common outlet 130 to prepare the micro fluidic device 130 for the pre-depletion steps.

[00048] The pre-depletion steps are performed as follows. A 1 mL syringe is filled with

1% BSA and attached to a 2G needle. The syringe/needle is attached to inlet tubing with 1% BSA until a small drop appears at the tip of the plug. The plug end is inserted into the common inlet 120 of the microfluidic device 110. The outlet tubing is plugged in. The 1% BSA is run through the common inlet 120 for 500 μL at 100 μL/min, thereby blocking the microfluidic device 110. After the 1% BSA block, the outlet/waste tubing is removed. The connection tubing is flushed with 1% BSA and attached to the common outlet 125. 1% BSA is flowed from the common inlet 120 through the micro-channels 115 and the common outlet 125 until a small drop appears at the end of the connection tubing. The microfluidic device 110 can be connected to other microfluidic devices via connection tubing. Alternatively, the common outlet 120 can be interfaced with the second micro-channel 130 to capture CTCs. [00049] Inlet tubing to the second micro-channel 130 can be 3" tygon tubing with needle tips at both ends, and 3" Teflon tubing attached. The block is run, as described previously, through the outlet of the second micro-channel 130. After blocking, the inlet tubing of the second micro-channel 130 is interfaced with the common outlet 125 of the micro fluidic device 110. The 1% BSA syringe is changed for a 1 mL syringe filled with the whole blood sample. The blood is pumped for 500 μL at 50 μL/min. After the blood is run, the microfluidic device 115 is detached from the second micro-channel 130 so that the device 115 and the micro-channel 130 are in parallel, not in series. The outlet tubing is placed back into the outlet of each of the device 115 and the micro-channel 130. The devices are washed individually with IX PBS for 500 μL at 100 μL/min. After the wash, 4% PFA or BD Cytofix/cytoperm is fixed for 500 μL at 100 μL/min.

[00050] Then the devices can be stained for specific markers including DAPI, CD45 and

Cytokeratin. Briefly, the devices can be subsequently washed with a solution of 0.9 mL of 0.2% Triton X-100 in PBS for 10 minutes at a flow rate of 50 μL/min to induce cellular permeability and allow for intracellular staining. To identify any bound lymphocytes, 0.9 mL of anti-CD45 stock solution (50 μL of antibody stock solution in 1 mL of PBS) can be passed through the chip at a flow rate of 50 μL/min and incubated for 60 minutes, followed by a PBS wash at 100 μL/min for 500 μL to remove excess antibody. To identify epithelial cells, 0.9 mL of anti- cytokeratin stock solution (50 μL of antibody stock solution in 1 mL of PBS) can be passed through the chip at μL/min and incubated for 60 minutes, followed by a PBS wash. If the above mentioned antibodies are not pre-conjugated with fluorroscent molecules, secondary antibodies (specific to primary antibody host) can be flown through the device for a total volume of 0.9 mL at 50 μL/min and incubated for 60 minutes. Finally, to permit the identification of cellular nuclei, 0.9 mL of DAPI solution (10 μl of DAPI reagent in 1 mL of DI water) can be passed through the chip at 50 μL/min incubated for 15 minutes followed by a PBS wash.

[00051] To store the device 115, the outlet tubing is sealed with tape after fixation or PBS wash. The device 115 is placed in a Petri dish with a balled, wet, cleaning cloth, for example, Kimwipe®, to the side of the devices. Several drops of PBS are dropped over the inlet and the outlet. The dish is sealed with parafilm and stored at 4 ⁰C. The cut corners of the device 115 are the outlet. The device 115 is symmetrical, and can consequently be functionalized from any end. Care should be taken to ensure that there is always a liquid-liquid interface and that no bubbles enter the micro-channels 115. To do so, the syringe should be flicked and inverted to remove bubbles. It should also be ensured that there is liquid on top of the device 115. The device 115 should preferably not dry out. One technique to prevent bubbles from entering the device during syringe/solution change can be to press gently on the device 115 near the inlet to cause liquid from the inside of the device to emerge. The aforementioned steps can be run for 500 μL at 100 μL/min, with blood and antibodies as the exception. These can be run at 25 μL/min for 500 μL.

[00052] The device 115 can be preserved after staining by making a plug loop which entails putting a needle tip on both ends of a 6" piece of tygon tubing, such as the tubing being used at the outlet. The syringe pump can be run from the common inlet 120 until fluid fills the outlet tubing and a drop appears at the end of the free needle tip. This can then be inserted in the place of the inlet tubing, thus making a loop. The looped device can be placed in a Petri dish with a small balled wet (for example, with PBS) cleaning cloth, for example, Kimwipe®, which should preferably not touch the device, and sealed with parafϊlm.

[00053] The depletion and capture described here can be done in a flow through fashion.

For enhanced pre-depletion of leukocytes, two pre-depletion microfluidic devices can be used in a row connected to the second micro-channel 130 as described with reference to figure 1. The whole blood sample 105 can flown at a constant flow rate through the microfluidic device 115 first and then into the second micro-channel 130. After processing typically 500 μL of blood, the microfluidic device 115 is disconnected from the second micro-channel 130, and can be washed with IX PBS. Subsequent to washing, the CTCs are fixed with 4% PFA for 20 minutes, permeablized with 0.2% Triton X-100 for 20 minutes for intracellular staining and DAPI. After staining, the excess antibodies are washed with IX PBS and the devices are imaged under fluorescent microscope.

[00054] Figure 6 is a table of cell populations, capture antibodies, and shear stresses to bind a cell population to a corresponding capture antibody. The shear stresses shown in figure 6 represent shear stresses for capturing leukocytes using CD45 antibody, neutrophils with CD66 antibody, and T-lymphocytes with CD2 antibody at corresponding volumetric flow rates. [00055] Figures 7 A and 7B show an image of a micro fluidic device to capture leukocytes and capture efficiencies of the device at different volumetric flow rates. Specifically, figure 7 A shows an image of the micro fluidic device. To determine the flow rate for enhanced capture on CD45 -coated micro-channels 115, whole blood collected from healthy volunteers was flown at three different flow rates, followed by a wash with IX PBS. After rinsing the excess blood and unattached cells, the cells captured on the surface of chamber are stained with DAPI. As shown in the figure 7B, the capture efficiency was determined by the cell density/mm² was high at the flow rate of 50 μL/min. This corresponds to a shear stress of approximately 0.82 dynes/cm².

[00056] The second micro-channel 130 can be functionalized to capture CTCs as follows.

The surface of the second micro-channel 130 can be functionalized with EpCAM antibodies using Avidin-Biotin chemistry. The surface of the micro-channel 130 can be modified with 4% (v/v) 3-mercaptopropyl trimethoxysilane in ethanol at room temperature for 45 min, then treated with the coupling agent N-y-maleimidobutyryloxy succinimide ester (GMBS, lμM) resulting in GMBS attachment to the micro-posts. Next, the micro-channel 130 can be treated with 10 μg/mL of Neutravidin at room temperature for 30 min leading to immobilization onto GMBS, and then flushed with PBS to remove excess Avidin. Finally, biotinylated EpCAM antibody at a concentration of 10 μg/mL in phosphate buffered solution (PBS) with 1% (w/v) BSA and 0.09% (w/v) sodium azide can be allowed to react for 15-30 minutes before washing with PBS. The micro-channel 130 can be air dried and stored at ambient temperature for up to three weeks until use.

[00057] Figure 8 shows a chart of non-specific binding of target cells in the micro- channels at varying volumetric flow rates. Since the number of CTCs present in blood circulation are so rare, loss of any CTCs in the process of pre-depletion was decreased. To determine the loss of target cells in the micro fluidic device 110, the whole blood was spiked with pre-labeled (with cell tracker orange) NSCLC cells H1650s at a concentration of 5000 cells/mL. As shown in figure 8, none of the H 1650 cells got captured in the micro-channels 115 of the micro fluidic device 110. At 30 μL/min flow rate, few cells were observed on the bottom of the micro-channels 115 - however the total number of cells that got non-specifically bound to the micro-channels 115 is less than 0.0001% of the total number of cells that went through the micro fluidic device 110. [00058] Figure 9 is a chart showing comparing non-specific binding per unit volume of whole blood sample in the presence of and absent pre-depletion. To study the effect of pre- depletion on the non-specific attachment of leukocytes on the second micro-channel 130, blood was collected from healthy volunteers. The blood was first introduced in the CD45 pre-depletion micro fluidic device 110 connected in series and then into the second micro-channel 115 which were all connected through in-line tubing. As shown in the figure 9, significant decrease in nonspecific binding per mL of pre-depleted whole blood, relative to non-specific binding in whole blood that was not pre-depleted, was observed.

[00059] The methods described here can result, for example, in the isolation of 50%, 60%,

70%, 80%, 90%, 95%, 99%, or 100% of the non-specific binding cells, for example, leukocytes, in a sample while retaining, for example, less than 20%, 10%, 5%, or 1% of the target cells. At least two variables can be manipulated to control the shear stress applied to the channel: the cross sectional area of the micro-channel and the fluid pressure applied to the micro fluidic device. Other factors may be manipulated to control the amount of shear stress necessary to allow binding of desired analytes and to prevent binding of undesired analytes, for example, the binding moiety employed and the density of the binding moiety in the channel.

[00060] Fluid pressure can be exerted by one of several techniques including syringe pumps, peristaltic pumps, vacuum. Methods for coupling pumps to devices are known in the art. The device may be configured for substantially constant shear stress in any given chamber or variable shear stress in a given chamber. Devices of the invention may be fabricated using techniques known in the art. The fabrication techniques employed will depend on the material used to make the device. Examples of fabrication techniques include molding, photolithography, electro forming, and machining. Exemplary materials include glass, polymers (e.g., polystyrene, silicones such as PDMS, epoxy, and urethanes), silicon and other semiconductors, and metals. Binding moieties may be attached to chambers using methods known in the art. The method employed will depend on the binding moiety and the material used to construct the device. Examples of attachment methods include non-specific adsorption to the surface, either of the binding moiety or a compound to which the binding moiety is attached or chemical binding, for example, through self assembled monolayers or silane chemistry. Devices of the invention may be combined with fluids, pumps, andlor detectors. Devices may also be combined with reagents, for example, lysis reagent, labeling reagents, and instructions for use, for example, for disease diagnosis.

[00061] 3-Mercaptopropyl trimethoxysilane was obtained from Gelest (Morrisville, PA).

Ethanol (200 proof), tissue culture flasks, a hemocytometer, serological pipettes, were purchased from Fisher Scientific (Fair Lawn, NJ). Fetal bovine serum (FBS) and 0.5 M ethylene diamine tetra acetic acid (EDTA) were purchased from Gibco (Grand Island, NY). Dimethyl sulfoxide (DMSO), sodium azide, lyophilized bovine serum albumin (BSA), and a glovebag for handling the moisture-sensitive silane were obtained from Sigma Chemical Co. (St. Louis, MO). The coupling agent GMBS (N-y-maleimidobutyryloxy succinimide ester), NHS-LC-LC-biotin (succinimidyl-6ø-[biotinamido]-6-hexanamido hexanoate), and fluorescein-conjugated NeutrAvidin were obtained from Pierce Biotechnology (Rockford, IL). Biotinylated mouse anti- human anti-EpCAM was obtained from R&D Systems (Minneapolis, MN). Human non-small- cell lung cancer line NCI-H1650, prostate cell line PC3-9, breast cancer cell line SKBr-3 and bladder cancer cell line T-24 were purchased from American Type Culture Collection (Manassas, VA), and RPMI- 1640 growth medium was purchased from Invitrogen Corporation. Orange [5- (and 6-)-(((4-chloromethyl)-benzoyl) amino) tetramethyl-rhodamine, CMTMR] and green [5-chloromethylfluorescein diacetate, CMFDA] cell tracker dyes were obtained from Molecular Probes (Eugene, OR). Anti-Cytokeratin PE (CAM 5.2, conjugated with phycoerythrin), CD45 FITC, the fluorescent nucleic acid dye nuclear dye 4',6-diamidino-2- phenylindole (DAPI) and 1OmL vacutainer tubes was purchased from BD Biosciences (San Jose, CA).

[00062] While this specification contains many specific implementation details, these should not be construed as limitations on the scope of any inventions or of what may be claimed, but rather as descriptions of features specific to particular embodiments of particular inventions. Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.

[00063] In alternative implementations, the target cells are CD4+ lymphocytes, and the whole blood sample is obtained from a patient at risk of developing AIDS. In such implementations, the microfluidic device including the multiple micro-channels includes a binding moiety for monocytes, for example, anti-CD 14, and the second micro-channel includes a binding moiety for CD4+ lymphocytes. One critical factor for accurate CD4 counting using this approach is the specificity of cell capture, which can be achieved with immobilized antibodies, which are further blocked with BSA to reduce non-specific binding (Arniji and Park. J. Biomater. Sci. Polym. Ed. 4:217 (1993)). Because CD4 is also expressed on monocytes, shear stress is used as a secondary selection step to exclude monocytes. CD4+ T lymphocytes and monocytes respond differently to shear stress on the functionalized device surface, as preferential binding of lymphocytes occurs in a window of 1-3 dynes/cm²; by contrast, monocytes bind at lower shear stresses.

[00064] Within the optimal lymphocyte-binding window (1-3 dynes/cm²), the shear force exerted on a cell 10 pm in diameter is -8-25 pN. This is of the same order as the binding force of a single antibody-antigen pair. (Harada et al. Langrnuir 16:708 (2000) and Hinterdorfer et al. Proc. Natl. Acad. Sci. U. S. A. 93:3477 (1996)). When the shear force is above this level, up to two orders of magnitude drop in cell adhesion is observed. This observation implies that when target cells come into contact with the surface, cell-substrate attachment is initiated by the formation of a single antibody- antigen interaction (Tissot et al. Biophys. J. 61 :204 (1992)), and high membrane antigen density will favor the opportunity of such interaction.

[00065] The table below provides exemplary cell populations, cell surface markers appropriate for the methods and devices described in this disclosure, and the corresponding shear stresses at which the indicated cells can be pre-depleted from a blood sample.

Claims

CLAIMSWe Claim:

1. A method for capturing target cells from whole blood, the method comprising: forming a first plurality of micro-channels on a micro fluidic device, the first plurality of micro-channels being parallel to each other and having a common inlet and a common outlet; configuring each micro-channel in the first plurality of micro-channels to bind leukocytes; coupling the first plurality of micro-channels with a second micro-channel configured to bind target cells; flowing a sample of whole blood including leukocytes and the target cells through the first plurality of micro-channels, wherein the first plurality of micro-channels bind the leukocytes such that the sample is depleted of leukocytes to form a depleted sample; and flowing the depleted sample through the common outlet and into the second micro- channel, wherein the second micro-channel binds the target cells.

2. The method of claim 1 , wherein configuring each micro-channel in the first plurality of micro-channels to bind leukocytes includes treating each micro-channel with CD2 configured to bind lymphocytes.

3. The method of claim 2, wherein flowing the sample of whole blood including leukocytes and the target cells through the first plurality of micro-channels further comprises flowing the sample of whole blood at a shear stress substantially equal to 1.75 dynes/cm² to bind the lymphocytes.

4. The method of claim 1 , wherein configuring each micro-channel in the first plurality of micro-channels to bind leukocytes includes treating each micro-channel with CD66 configured to bind neutrophils.

5. The method of claim 2, wherein flowing the sample of whole blood including leukocytes and the target cells through the first plurality of micro-channels further comprises flowing the sample of whole blood at a shear stress substantially equal to 0.45 dynes/cm² to bind the neutrophils.

6. The method of claim 1 , wherein configuring each micro-channel in the first plurality of micro-channels to bind leukocytes includes treating each micro-channel with CD45 configured to bind leukocytes.

7. The method of claim 6, wherein flowing the sample of whole blood including leukocytes and the target cells through the first plurality of micro-channels further comprises flowing the sample of whole blood at a shear stress substantially equal to 0.82 dynes/cm² to bind the leukocytes.

8. The method of claim 1, wherein the target cells include circulating tumor cells, the method further comprising functionalizing surfaces of the second micro-channel with EpCAM antibodies using Avidin-Biotin chemistry such that the functionalized surfaces bind the circulating tumor cells.

9. The method of claim 1, further comprising: forming a second plurality of micro-channels; configuring each micro-channel in the second plurality of micro-channels to bind leukocytes; coupling the common outlet of the first plurality of micro-channels to a common inlet of the second plurality of micro-channels; coupling a common outlet of the second plurality of micro-channels to the second micro- channel; and flowing the whole blood sample through the first plurality of micro-channels and the second plurality of micro-channels to form the depleted sample.

10. The method of claim 1, further comprising forming the second micro-channel on a separate microfluidic device.

11. The method of claim 1 , wherein coupling the first plurality of micro-channels with the second micro-channel comprises providing a fluid connection between the common outlet of the first plurality of micro-channels to an inlet of the second micro-channel using a capillary tube.

12. The method of claim 1, further comprising flowing IX PBS through the first plurality of micro-channels after flowing the sample of whole blood, wherein the IX PBS unbinds the leukocytes from the first plurality of micro-channels.

13. An apparatus for capturing target cells from whole blood, the apparatus comprising: a microfluidic device including a first plurality of parallel micro-channels having a common inlet and a common outlet, the first plurality of micro-channels treated with a first binding moiety such that the first plurality of micro-channels bind leukocytes; a second micro-channel including an inlet and an outlet, wherein the second micro- channel is treated with a second binding moiety such that the second micro-channel binds target cells; and an interface coupling the common outlet of the first plurality to the inlet of the second micro-channel, wherein a whole blood sample including leukocytes and the target cells that is flowed into the common inlet of the first plurality flows through the first plurality of parallel micro-channels, through the common outlet of the first plurality, and into the inlet of the second micro-channel through the interface, such that the whole blood sample flowing through the common outlet of the first plurality is depleted of leukocytes.

14. The apparatus of claim 13, wherein the first plurality of micro-channels consists of sixteen micro-channels.

15. The apparatus of 13, wherein each of the first plurality of micro-channels is 50 μm deep, 4 mm wide, and 14 mm long.

16. The apparatus of claim 13, wherein the first binding moiety is CD66, and wherein dimensions of each of the first plurality of micro-channels is selected to exert a shear stress of 0.45 dynes/cm² on the whole blood sample at a whole blood sample flow rate of 30 μL/min.

17. The apparatus of claim 13, wherein the first binding moiety is CD2, and wherein dimensions of each of the first plurality of micro-channels is selected to exert a shear stress of 1.75 dynes/cm² on the whole blood sample at a whole blood sample flow rate of 100 μL/min.

18. The apparatus of claim 13, wherein the first binding moiety is CD45, and wherein dimensions of each of the first plurality of micro-channels is selected to exert a shear stress of 0.82 dynes/cm² on the whole blood sample at a whole blood sample flow rate of 50 μL/min.

19. The apparatus of claim 13, wherein the second micro-channel is formed on the micro fluidic device.

20. The apparatus of claim 13, wherein the second micro-channel is formed on a second microfluidic device that is separate from the microfluidic device, and wherein the target cells are circulating tumor cells.

21. The apparatus of claim 20, wherein the second microfluidic device is 25 mm x 66 mm and wherein the second microfluidic device has an active capture area of 19 mm x 51 mm.

22. The apparatus of claim 21, wherein the active capture area includes a plurality of micro- posts, and wherein the second binding moiety includes EpCAM antibodies, wherein each micro- post is treated with EpCAM antibodies using Avidin-Biotin chemistry such that the micro-posts bind the circulating tumor cells.

23. The apparatus of claim 22, wherein the capture area includes 78,000 micro-posts, each micro-post being 100 μm tall and 100 μm in diameter, wherein the plurality of micro-posts are arranged in an equilateral triangular array with a 50 μm gap between each micro-post.

24. The apparatus of claim 13, wherein the interface is a capillary tube.

25. The apparatus of claim 13, wherein the interface is detachable from the common outlet of the first plurality and the inlet.