Abstract:
A particle counter for analyzing blood has features which provide for automatic operation and preferably, also provide for portable use in a low resource setting. In a preferred embodiment, preferred embodiment, the device is used to obtain CD4 counts for AIDS diagnosis.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application claims priority to U.S. Provisional Applications 60/947,384 and 60/947,345, filed 29 Jun. 2007 entitled “Microfluidic Device for Counting Cells” hereby incorporated by reference in their entireties herein. 
     
    
     BACKGROUND 
       [0002]    Practical HIV diagnostics are urgently needed in resource-limited settings. While HIV infection can be diagnosed using simple, rapid, lateral flow immunoassays, HIV disease staging and treatment monitoring require accurate counting of a particular white blood cell subset, the CD4(+) T lymphocyte. 
         [0003]    Current systems for providing this function are expensive, technically demanding and/or time-consuming. For example, CD4 counts may be obtained by conventional flow cytometry. The method requires a slow flow rate to capture the cells on the surface of the channel, followed by a quick wash. This is not easy to implement in a point-of-care device, where the fluid actuation technique must be fairly simple. They require a microscope and a camera (or a person) to count the captured cells. This method is suitable for a lab environment, not point of care. 
       SUMMARY 
       [0004]    According to an embodiment, the invention is a portable microfluidic device for counting CD4+ T-cells. In a method embodiment, the device is used to count CD4+ T-cells and the information from the counting is used as a key diagnostic criterion for determining whether to administer antiretroviral therapy to HIV-infected patients. In the device embodiment, a disposable microfluidic cassette contains densely packed beads for capturing T-cells. A portable, self-powered instrument operatively associated with the microfluidic cassette reads and displays the result of the counting. Preferably, the device employs bead-based microfluidics to enhance surface area for cell capture. For example, in an embodiment, a “double-dam” design is employed which is compatible with whole blood. Preferably, a small pump is employed to permit complete compactness and low power requirement. Also, preferably, the device uses a portable detection device based on an absorbance/chemiluminescence reader. In a particularly preferred embodiment, the detector is powered by a solar batter, which can, for example facilitate its use in point-of-care settings in the developed countries, as well as developing countries. 
         [0005]    In a particular variation, the device uses cell affinity chromatography operated under differential shear flow to specifically isolate CD4(+) T lymphocytes. In an embodiment, the device may be effective with a direct processing of 10 microliters of unprocessed, unlabeled whole blood. CD4 counts are, in this embodiment, obtained under an optical microscope in a rapid, simple and label-free fashion. For example, the embodiments are effective for a range of absolute CD4 counts (R(2)=0.93) over which the embodiments are operable. This CD4 counting microdevice can be used for simple, rapid and affordable CD4 counting in point-ofcare and resource-limited settings. 
         [0006]    In a preferred configuration, packed beads are provided in a microchannel to enhance capture surface area which increases the cell-capture efficiency to enhance the final signal, and allows constant flow, permitting the use of a small pump, preferably peristaltic and preferably powered by a battery. Preferably, capture and detection are done in the same location. Optics are employed that automatically reads the signal either by absorbance or chemiluminescence or some other suitable technique. The optical components are preferably portable and as such, are, in the preferred embodiment, powered by battery including the detection portion, the pumping portion, and the microfluidic channel chip. Preferably, rechargeable batteries are used. 
         [0007]    A portable blood component analysis device has a microfluidic unit with a sample chamber defined in a first longitudinal channel having a first major axis and a second longitudinal channel, having a second major axis, in communication with the first longitudinal channel with the second major axis crossing the first major axis; the sample chamber having a surface augmentation features with biospecific surface configured to capture particles from blood; an analyzer component having a signal detector and a receiving slot configured to receive the microfluidic unit and align the sample chamber with the signal detector. Preferably, the first and second major are substantially perpendicular. Preferably, the analyzer contains a pump configured to generate a vacuum of at least 10 kPa. Preferably, the surface augmentation features include a packed bead bed with glass beads. Preferably, the surface augmentation features include a packed bead bed with glass beads with a diameter between 50 and 100 μm and the first longitudinal channel contains a narrow portion with a minimum dimension of less than the diameter of the glass beads. Preferably, the device further includes a pump, a controller and a solar power source connected to power the pump and controller. Preferably, the microfluidic unit includes a tube containing at least one, at least one wash, and at least one air bubble, the tube being reversibly sealed at both ends and configured to connect with the second longitudinal channel. Preferably, the device further includes a pump, a controller and a solar power source connected to power the pump and controller, wherein the microfluidic unit includes a tube containing at least one, at least one wash, and at least one air bubble, the tube being reversibly sealed at both ends and configured to connect with the second longitudinal channel and wherein the controller is configured automatically to pump fluids from the tube through the sample chamber and through the second longitudinal channel. In any of these devices, the blood particles detected may include CD4 cells. Preferably, a battery is included. Preferably, a controller receives a signal from the signal detector and the signal is a light intensity, the controller being configured to derive a cell count from a light intensity signal without imaging. Preferably, the controller is configured to derive the cell count from a calibration lookup table correlating cell count against total light intensity from the sample chamber. 
         [0008]    Another portable blood component analysis device has a microfluidic unit with a sample chamber; the sample chamber having a surface augmentation features with biospecific surface configured to capture particles from blood. an analyzer component having a signal detector and a receiving slot configured to receive the microfluidic unit and align the sample chamber with the signal detector; Preferably, the surface augmentation features include a packed bead bed with glass beads. The device claim  13 , wherein the analyzer contains a pump configured to generate a vacuum of at least 10 kPa; Preferably, the surface augmentation features include a packed bead bed with glass beads with a diameter between 50 and 100 μm and the first longitudinal channel contains a narrow portion with a minimum dimension of less than the diameter of the glass beads. Preferably, the device includes a controller and a solar power source connected to power the pump and controller. Preferably, the microfluidic unit includes a tube containing at least one, at least one wash, and at least one air bubble, the tube being reversibly sealed at both ends and configured to connect with the second longitudinal channel. Preferably, the device has a pump, a controller and a solar power source connected to power the pump and controller, wherein the microfluidic unit includes a tube containing at least one, at least one wash, and at least one air bubble, the tube being reversibly sealed at both ends and configured to connect with the second longitudinal channel and wherein the controller is configured automatically to pump fluids from the tube through the sample chamber and through the second longitudinal channel. Preferably, the blood particles include CD4 cells. Preferably, the device has a battery powered by solar power source. Preferably, the device has a controller and the signal detector is a light intensity detector and the controller is configured to derive a cell count from a light intensity signal without imaging. Preferably, the controller is configured to derive the cell count from a calibration lookup table correlating cell count against total light intensity from the sample chamber. Preferably, the device has a display configured to output a cell count. Preferably, the device has a controller and the signal detector is a light intensity detector and the controller is configured to derive a cell count from a light intensity signal without imaging. The device of claim  23 , wherein the controller is configured to derive the cell count from a calibration lookup table correlating cell count against total light intensity from the sample chamber. Preferably, the device includes a pump, a controller and a solar power source connected to power the pump and controller, wherein the microfluidic unit includes a tube containing at least one, at least one wash, and an a chemiluminscence activator, the tube being reversibly sealed at both ends and configured to connect with the second longitudinal channel and wherein the controller is configured automatically to pump fluids from the tube through the sample chamber and through the second longitudinal channel. Preferably, the pump is controlled to vary a pumping rate depending on a sample or reagent passing through the sample chamber. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0009]      FIG. 1  shows a first embodiment of a sample analyzer. 
           [0010]      FIGS. 2A and 2B  show a second embodiment of a sample analyzer. 
           [0011]      FIG. 3  shows a multiple-chamber sample analyzer showing various features. 
           [0012]      FIGS. 4A ,  4 B, and  4 C illustrate embodiments of portable sample analyzer machines. 
       
    
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
       [0013]    Referring to  FIG. 1 , a T-shaped microfluidic device  100  has a sample channel  100 S for sample fluid and reagents and a loading channel  100 L for loading particles that providing surface augmentation, such as glass or polystyrene beads  106 . The particles preferably carry immobilized biospecific molecules or other reactive substances on their surfaces. An inlet channel  101  and an outlet channel  102  are used to convey a sample fluid, such as blood, and reagents and washes through the sample channel  100 S. The loading channel  100 L is used to fill a chamber  104  with the surface augmentation particles to form a packed bed within the chamber  104 . The beads  106  are trapped in a cage made up of microposts  108  defining sample inlet, sample outlet fences  110 ,  112 , and  114 . The beads may be suspended in a fluid and conveyed through a loading port  107  along with the fluid suspending them into the chamber  104  while permitting the fluid to drain through gaps between the posts  108 . The beads may be loaded in by flowing them in a suspension by pumping a fluid into the bed  104  area and drawing excess carrying fluid out through a fence  114  trapping the beads  106  in the bed  105  and packing them with a repeatable density. 
         [0014]    Preferably, the gap between the posts is smaller than the breads. The Posts  108  can be many times the size of the beads. The beads do not have to be a constant size, but preferably they are a consistent size to help ensure a packing arrangement with a large and predictable void volume. If desired, a step may be added, for example, to coat the bead particles with a stabilizing or protecting material. 
         [0015]    Although not shown in the drawing, the chamber  104  is enclosed at the top and bottom. The entire structure can be made using lithographic techniques that are well known. Once packed with the surface augmenting particles, loading channel  100 L can be permanently sealed at both ends and the sample channel  1005  temporarily sealed for storage and/or shipping. The device  100  may be combined with multiple such devices  100  on a single piece of substrate, each with a bed having a different immobilized biospecific substances to permit detection of different sample fluid components. In use, the sample channel  100 L is unsealed such that fluid can enter and leave chamber  104  through the inlet and outlet channels  101  and  102 . The drawing does not show the beads  106  in place in the bead bed  104 . 
         [0016]    Referring to  FIGS. 2A and 2B , an array of sample channels  231 ,  232 ,  233 , and  234 , each having a sample chamber  218 , is formed in a substrate  201 . The sample chambers  218  are functionally similar to the chamber  104  of  FIG. 1 , except that in the present embodiment, narrow channels  216  are used to retain the surface augmentation particles  106 , for example, beads. The beads are loaded into loading ports  202  (as indicated by arrow  237 —only one port  202  is labeled to avoid crowding the drawing, but the others are similarly configured). The beads are held back by the narrow channels  216  and the fluid carrying the beads exits as shown by the arrow  239  out of an exit port  204 . Using these structures, beads with different immobilized substances can be loaded in each sample chamber  218 . Once the sample chambers  218  are packed, the ports  202  and  204  are sealed and the device  200  can be used or stored and/or shipped. In other respects, the device  200  is similar to that described with reference to  FIG. 1 . 
         [0017]    In use, the sample, washes, and reagents (collectively indicated by arrow  235 ) are loaded through sample inlet ports  212  and spent fluids recovered or disposed of through sample outlet port  206  (arrow  237 ). As described below, sample channels  210  convey a fluid train (the sample, washes, and reagents) serially through each of the sample chambers. Preferably, the fluid train is drawn through the device  200  using a vacuum pump regulated to maintain a pressure that provides desired shear rate which has been determined to remove particles that do not bind with the beads (more specifically, the active surface of the bead particles) and permit those particles (such as cells) that do bind to remain in the sample chambers  218 . In certain embodiments, it will preferably be determined the range of shear rates that provide the desired discrimination behavior. 
         [0018]      FIG. 2B  illustrate, as viewed from the top, a single sample chamber  218  with beads  240  packed within. The sample channel  210  permits the train of sample fluid and reagents to pass through. Preferably, the sample line has a size that prevents the migration of substantial quantities of beads  240  from the sample chamber  218 . In an embodiment, the sample channel width is selected to be slightly smaller than the coated beads. 
         [0019]    As will be described below, sample fluid particles in sample fluid, such as CD4 cells (particles) in blood (sample fluid) flow into the sample chamber followed by a wash, followed by a fluid carrying free signal molecules, followed by a wash, followed by a fluid carrying a free signal amplifier molecule. In each case, a different shear rate may be desirable to ensure that only the reactive particles or molecules are left behind and such that the shear rate is not so high as to interfere with the ability of particles to attach ultimately to the beads or substances thereon. The shear rate may vary depending on the component of the assay, for example, when a wash is applied versus when a reagent is applied. Shear rate may vary for different reagents as well. Preferably, an automated device, as described below, is provided with a controller to vary the total vacuum to vary the shear rate accordingly. This may be synchronized to the stage of the assay by detecting a property of the fluid train (e.g. detecting air bubbles that isolate the fluids from each other), by time after start of the vacuum pump, by detecting momentary changes in the vacuum pressure caused by passage of different fluids, such as air, into the beds, or other means. 
         [0020]    Referring to  FIG. 3 , beads  240  configured for capturing the target cells (such as by anti-target cell antibody) are packed in a bed  218  in an analyzer device  300 . The device has tubing  312  with connectors  356  that connect to sample fluid train  351  and a vacuum pump  306 . The sample fluid train may be held in a long tube  307  which is sealed with rupturable seals  350 . The rupturable seals may be configured to rupture upon application of a vacuum or by being pierced automatically by a sharp edge (not shown) when installed in a portable device. After the sample fluid train passes through the device  300 , a signal detector  302  may detect light caused by a reaction, for example, chemiluminescence or fluorescence, In the latter case, a light source, such as photodiode  304  may be provided. In a preferred embodiment, the number of cells captured in the sample chamber is the quantity of interest and preferably the system is configured such that the total amount of light captured by the detector indicates the number of cells according to a predetermined calibration curve stored in a portable controller and the cell count or other indication displayed automatically by the device. 
         [0021]    The train of fluid  310  including sample fluid  320 , such as whole blood conveyed through an inlet channel  312  to the bed  218 . The sample fluid train may include further fluids, each separated from each other, as appropriate in view of their mutual activity, by air or gas bubbles  340 . The fluid train may also include washes  322 ,  328 , fluids carrying intermediate materials such as antibodies that bind signal molecules, fluids carrying signal molecules  324 , developers and, fluids carrying signal amplifiers  326 , etc. For another example, the sample fluid train may include substance that causes chemoluminescence. 
         [0022]    In a preferred embodiment, the target that reacts with the reactive substance on the beads is lymphocytes (for example CD4 cells) carried in the sample fluid, blood. In such embodiment, is followed by a wash and then a target cell antibody (the primary antibody). In the principal embodiment, this is a CD4 cell primary antibody; i.e., an antibody that recognizes a CD4 cell. Following that, a quantity of a detectable (labeled) secondary antibody that recognizes the CD4 cell antibody is conveyed through the inlet channel and binds or associates with the primary antibody. A developer may be conveyed as well to enhance or permit detection. 
         [0023]    As mentioned, to detect the quantity of target cells retained in the sample chambers, the device preferably measures the strength of an optical signal. Examples included fluorescence, label (e.g. silver) absorbance, and chemiluminescence. Techniques which are known in the prior art, may be employed. A small electronic (charge-coupled device; “CCD”) camera, microscope, or other suitable photodetector device can be employed with suitable optics and filtering to provide an indicator signal. The total magnitude of the light signal may be compared to a standard to quantify the count of cells. 
         [0024]    In an exemplary embodiment, the embodiments employ dimensions that are determined to permit the close packing in a single layer while permitting sufficient flow of sample fluid and reagents without clogging. The dimensions of the embodiment of  FIG. 1  may include 90 μm-diameter microposts  108  spaced apart a distance smaller than the beads  108 , for example, spaced 25 μm apart to trap them. The beads are 40 μm-diameter and the depth (dimension parallel to the axes of the microposts  108 ) of the channel defined by the bed  104  is 75 μm-tall channels. In an exemplary embodiment, the bed  104  is 2 mm by 2 mm. The void fraction of the packed bed  104  may be chosen such that cells clear the column after a few washes. 
         [0025]    Referring to  FIG. 4A , a schematic of a portable analyzer  400  has a power source  404 , for example, a solar cell or hand generator. A microprocessor based microcontroller (XTL)  402  controls the unit and drives a pump, (optionally, if used) a light source (e.g., laser diode) or other exciter  412 , photodetector  410 , and a display  408 , preferably an LCD. Inputs from a user may be provided by suitable actuators such as membrane switches to allow a user to operate the device. Switches,  418  and  420  may be limited to power on and run functions to make the unit fully automated and simple to operate. A housing  422  is preferably provided to form a unitary portable device. 
         [0026]    Referring to  FIG. 4B , a more detailed embodiment of a portable analyzer  450  has a vacuum pump  462  to generate a vacuum for drawing sample and reagents through a microfluidic chip  494  as discussed above. A pressure sensor  471  detects a pressure signal via a pressure chamber  464  which smooths out pressure pulses, indicating the vacuum output of the vacuum pump. Another pressure sensor  470  detects a pressure signal via a pressure chamber  468  which also smooths out pressure pulses, the latter signal indicating the regulated vacuum pressure determined by a pressure regulator  466 . The pressure signals are applied to a microcontroller  488  as are signals from user control actuators  472 , and photodetector signals from one or more photodetectors  476  amplified by an amplifier  480 . The signal from the photodetectors  476  may are preferably sampled and converted to digital data and processed for analysis to permit results to be displayed on a display  482 . Further output devices may include an indicator annunciator  484  and/or an indicator lamp  478 . The latter may be used, for example, to indicate the completion of an analysis and the status of the device  450 , respectively, for example. 
         [0027]    Referring to  FIG. 4C , in a preferred embodiment, a microfluidic chip  604  with reagent cache  602  (tube filled with reagent and having a sample port  603 ) is configured to be received in a recess  606  of an analyzer  608  to form a configuration as described with reference to  FIG. 4A  or  4 B. In the present embodiment, a swing-out front panel has a solar charger  614 . Control buttons such membrane switches (not shown) may be provided on the housing. A circuit board (not shown) carrying an LCD display  615  may be carried on the front panel. The microfluidic channel “chip”  604  (and fluid train cache  602 ) may be provided in the form of a replaceable component which is used once for each measurement and discarded. In a following layer a circuit board carries a microcontroller and photodetector (or camera or other sensor) positioned to detect light from the bead bed. Batteries and a pump may be carried in a lower chassis. The entire structure shown can be packaged in a hinged device which may be opened to insert the microfluidic chip while permitting access for the drawing of a sample fluid (and any detection or development agents required). 
         [0028]    In an exemplary embodiment, the laser diode may, for example, have a wavelength of 654 nm. The device may have an alignment slot, which is exposed when opened, to permit the proper location of the microfluidic chip. It may also have a Polydimethylsiloxane window to protect the detector and/or the light source. In an embodiment, the photodetector records changes in absorbance, for example due to opacity of a developing silver film. In another embodiment, the photodetector detects light emitted by captured sample material and labels, e.g., by fluorescence. Preferably, results are displayed digitally to minimize subjective interpretation by the user. 
         [0029]    To confirm the above configuration, a passive adsorption of antibodies onto beads was tested. The capture of 3T3 fibroblasts on the beads (coated with human fibronectin), and CD4 T-cells (using anti-CD4 antibodies) has been demonstrated by experiment. An ability to capture both fibroblasts and lymphocytes with this method ( FIG. 2A ), has been confirmed. The use of covalent chemistry has been investigated, using carboxylated beads and the carbodiimide coupling agent EDC. Beads coated with anti-CD4 successfully captured T-cells from whole blood of AIDS patients, by DIC ( FIG. 2B ) and fluorescence microscopy ( FIG. 2C ). Among commercially available antibodies, it was found that only a small number have strong affinity for CD4 and can be easily coated onto surfaces. Using this set of antibodies, we will finish a signal-to-background optimization by July 31 as scheduled. 
         [0030]    To prepare glass beads, the following methodology was experimentally confirmed as suitable for use in the above embodiments. First, beads having a diameter of about 50 μm were subjected to 2N nitric acid wash for 1 hour (to clean the glass beads). Next, the beads were silanized with 10% APTES (an aminosilane) overnight at room temperature. The beads were washed with toluene and acetone, and dried in an oven at  60 C for 2 hr. Then the beads were reacted with 2.5% glutaraldehyde (to crosslink terminal—NH2 on APTES with primary amines on proteins added later) for 1 hour at room temperature and subsequently washed with distilled water. The next step was to couple 10 μg/ml of protein G onto silanized glass surface (to orient anti-CD4+ antibody with F(ab) towards mobile phase, and to act as additional spacer). The next step was to wash 3 times with phosphate buffer saline solution and couple 10 μg/ml of mouse-anti-human CD4+ antibody onto protein-G coated glass surface. Specific binding of fluorescent antibodies was demonstrated, using bovine serum albumin (BSA) as a negative control to block binding of fluorescent secondary antibody markers (AF-488 goat anti-mouse antibodies). 
         [0031]    It has been experimentally determined that beads of a certain minimum size are preferred to reduce clogging by cell particles. In balancing the size against the reduction in surface, the preferred size range has been determined to be in the range of 50-100 and preferably about 75-80 μm diameter. An exemplary shear rate determined to provide reliable discrimination is 10-20 dyn/cm 2 .