PATENT DOCUMENT

Abstract:
An apparatus having scattering and multi-color fluorescence detecting, analyzing and identification capabilities of blood or other fluids of interest. The sample to be tested may be entered in a disposable microfluidic cartridge which in turn is insertable in a hand-holdable or implantable miniaturized and portable cytometer instrument. This cytometer has significant application in biological warfare agent detection, hematology and other clinical and research fields.

Full Description:
This Patent Application is a continuation-in-part of U.S. patent application Ser. No. 09/630,924, filed Aug. 2, 2000 now U.S. Pat. No. 6,597,438, and claims the benefit thereof. 
     CROSS-REFERENCE TO RELATED CO-PENDING APPLICATIONS 
     This Application is related to co-pending U.S. patent application Ser. No. 09/630,927 to Cabuz et al., filed Aug. 2, 2000, and entitled “Optical Detection System for Flow Cytometry”; U.S. patent application Ser. No. 09/630,924, to Cabuz et al., filed Aug. 2, 2000, and entitled “Portable Flow Cytometer”; U.S. patent application Ser. No. 09/630,923, to Cabuz et al., filed Aug. 2, 2000, and entitled “Fluid Driving System for Flow Cytometry”; U.S. patent application Ser. No. 09/896,230, filed Jun. 29, 2001, to Fritz, and entitled “Optical Detection System for Flow Cytometry”; and U.S. patent application Ser. No. 09/404,560, to Cabuz et al., filed Sep. 23, 1999, and entitled “Addressable Valve Arrays for Proportional Pressure or Flow Control”, all of which are incorporated herein by reference. 
    
    
     BACKGROUND 
     The present invention relates generally to flow cytometers. More particularly, the present invention relates to portable flow cytometers that sense optical properties of microscopic biological particles or components in a flow stream. 
     Flow cytometry is a technique that is used to determine certain physical and chemical properties of microscopic biological particles or components by sensing certain optical properties of the particles or components. To do so, for instance, the particles are arranged in single file using hydrodynamic focusing within a sheath fluid. The particles are then individually interrogated by a light beam. Each particle scatters the light beam and produces a scatter profile. The scatter profile is often identified by measuring the light intensity at different scatter angles. Certain physical and/or chemical properties of each particle can then be determined from the scatter profile. 
     Flow cytometry is currently used in a wide variety of applications including hematology, immunology, genetics, food science, pharmacology, microbiology, parasitology and oncology, to name a few. A limitation of many commercially available flow cytometer systems is that they are relatively large bench top instruments that must remain in a central laboratory environment. Accordingly, the use of such flow cytometers is often not available in remote locations or for continuous hematological monitoring. 
     SUMMARY 
     The present invention overcomes many of the disadvantages of the prior art by providing a highly miniaturized portable and wearable cytometer that can be used at remote locations, such as at home or in the field. Such a flow cytometer may help improve healthcare of patients by providing detailed individual hematological evaluation and uncovering statistical trends. By detecting an infection early, the infection may be more readily treatable. 
     In military applications, the portable miniaturized cytometer of the present invention may help save lives by providing early detection of infection due to biological agents. It is known that expanded activity in the biological sciences has increased the probability of accidental exposure to dangerous biological agents. The ease of manufacturing such agents also raises a serious threat to their use by terrorists, regional powers or developing third world nations. The lack of safeguards in international agreements outlawing biological warfare, and compelling evidence that those agreements may have been violated, reinforces the need for a strong capability for biological defense. Pre-exposure detection of pathogen agents, as well as post-exposure detection of incipient infections may be used cooperatively to ensure efficient protection during biological warfare. 
     As part of the body&#39;s natural defense against antigens, the white blood cell count increases at the onset of infection. There are several types of white blood cells including neutrophils, lymphocytes, monocytes, eosinophils and basofils. Lymphocytes create antibodies that attack the invaders and mark them for destruction by the neutrophils and macrophages. In an individual without chronic diseases (such as tuberculosis or cancer), an increase in the percentage of lymphocytes in the overall white cell count is an indication of a viral infection. On the other side, an increase in the percentage of the neutrophils is an indication of a developing bacterial infection. Through counting of neutrophils and lymphocytes, a clear infection warning can be issued with differentiation between viral or bacterial causes. 
     The first clinical symptoms of infection from some bacterial agents such as bacillus anthrax appear after one to six days. In 99% of the cases, patients showing symptoms from anthrax cannot be treated, and will most likely die. However, if treatment is given before the first symptoms appear, most patients can be successfully treated. Accordingly, it would be highly desirable to provide an early alert and potential therapeutic intervention for hematologic abnormalities before symptoms occur. In many cases, such an early alert and treatment may greatly improve the outcome for many patients. 
     In an illustrative example of the present invention, a portable miniaturized cytometer is provided for identifying and/or counting selected particles in a fluid sample such as a blood sample. One illustrative miniaturized portable cytometer includes a fluid receiver for receiving the fluid sample. One or more reservoirs are provided for storing supporting fluids such as lyse and sheath fluids. For many commercial flow cytometer systems, a precision fluid driving system is used for providing precise pressures to the fluids. A limitation of this approach is that precision fluid driving systems can be bulky, complex and may require significant power. 
     To avoid many of these limitations, an illustrative example uses a non-precision fluid driver that is controlled by a closed loop feedback path. The non-precision fluid driver is coupled to the fluid receiver and the various supporting fluid reservoirs, and applies separate pressures to the sample fluid and the supporting fluids. To control the velocity of the sample fluid and the supporting fluids, one or more valves are coupled to the fluid driver. The valves are used to regulate the non-precision pressures that are applied to the sample fluid and the supporting fluids by the non-precision fluid driver. 
     To complete the feedback loop, flow sensors are provided downstream of the fluid driver to measure the fluid velocity of the sample fluid and the supporting fluids. A controller or processor receives the signals from the flow sensors, and adjusts the appropriate valves so that the desired fluid velocities of the sample fluid and supporting fluids are achieved. The flow sensors are preferably thermal anemometer type flow sensors. 
     In one illustrative example, the non-precision fluid driver is manually powered. A manually powered fluid driver may include, for example, a bulb with check valve or a plunger. In either case, the manually generated pressure is preferably provided to a first pressure chamber. A first valve is then provided for controllably releasing the pressure in the first pressure chamber to a second pressure chamber. A second valve may be provided in the second pressure chamber for controllably venting the pressure in the second pressure chamber. The controller opens the first valve when the fluid flow in the downstream fluid stream drops below a first predetermined value and opens the second valve when the fluid flow in the downstream fluid stream increases above a second predetermined value. Each valve is preferably an array of electrostatically actuated microvalves that are individually addressable and controllable. 
     The controlled sample fluid and supporting fluids are provided to a fluidic circuit. The fluidic circuit performs hydrodynamic focusing, which causes the desired particles to fall into single file along a core stream surrounded by a sheath fluid. One or more light sources or light source arrangements provide light through the flow stream, and one or more light detectors or light detector arrangements detect the scatter profile and fluorescence of the particles in the flow stream. An arrangement may have one or more light sources and/or one or more light detectors. An arrangement may include a single optical device or element or an array of such items. A processing block uses the output signals from the light detectors to identify and/or count selected particles in the core stream. 
     The miniaturized portable cytometer may be provided in a housing sufficiently small to be appropriately and comfortably “wearable” on a person. In one illustrative example of the invention, the housing is sized similar to a wrist watch. The wearable housing may include, for example, a base, a cover, and a hinge that secures the base to the cover. The non-precision fluid driver and regulating valves may be incorporated into the cover, while the fluid reservoirs, flow sensors and fluidic circuit may be incorporated into a removable cartridge that is inserted into the housing. Preferably, the fluidic circuit dilutes the blood sample, performs red cell lysing, and performs hydrodynamic focusing for flow and core stream formation. The light sources are preferably situated in either the base or the cover, and aligned with the flow stream of the removable cartridge. The light detectors are preferably provided generally opposite the light sources. The processor and batteries may be provided in either the base or the cover of the housing. 
     The light source may include one or a linear array of first light sources along a first light source axis. The first light source axis may be rotated relative to the central axis of the flow stream. A lens may be provided adjacent each light source to focus the light at the particles in the core stream. A detector or set of light detectors may then be placed in-line with the light source or each of the light sources. Such an arrangement can be used to determine, for example, the alignment and width of the core stream within the flow stream. If the core stream of particles is not in proper alignment, the controller can adjust the fluid velocity of the sample fluid or one of the supporting fluids to bring the core stream into alignment. The light detector or set of light detectors may also be used to detect the velocity and size of each particle, as well as the number of particles. 
     Another light source or set of the light sources may be provided along second light source axis. A lens may be provided adjacent each light source to focus the light at the particles in the core stream. A second detector or set of light detectors may then be placed on either side of the in-line position of each light source for measuring the small angle scattering (SALS) produced by selected particles in the flow stream. 
     The second light source or set of light sources may also be used in conjunction with the first set of light sources to determine the time-of-flight or velocity of the particles in the flow stream. By knowing the velocity of the particles, small variations in the flow rate caused by the fluid driver can be minimized or removed by the controller. 
     A third light source or set of light sources may be provided along a third light source axis. A lens may be provided adjacent each light source to provide collimated light to the flow stream. An annular light detector or detectors may then be placed opposite the light source or light sources for measuring the forward angle scattering (FALS) produced by the selected particles in the flow stream. Each of the first, second and third light sources or sets of light sources may include an array of lasers such as vertical cavity surface emitting lasers (VCSELs) fabricated on a common substrate. Each of the first, second and third detectors or sets of light detectors may include a photo detector or an array of photo detectors such as p-i-n photodiodes, GaAs photodiodes with integrated FET circuits, resonant cavity photo detectors (RCPDs), or any other suitable light detectors. 
     The selected particles are preferably neutrophils and/or lymphocytes white blood cells. By examining the scatter profile of each particle, the miniaturized portable cytometer of the present invention identifies and counts the neutrophils and lymphocytes in a blood sample, and provides a clear infection warning with differentiation between viral and bacterial causes. 
     Another part of the invention uses of fluorescence to further identify and analyze various white cells. Antibodies may be associated with particular white blood cells. The antibodies have markers or tags attached to them. These white blood cells may be impinged with light which causes their associated markers or tags to fluoresce and emit light. The light may be collected, filtered as needed, and directed to one or more photo detectors. This detection may be used to identify and monitor specific subclasses of white cells and blood-based proteins, among other things. 
     In sum, this miniaturized portable cytometer has two optical detection subsystems—scattering and fluorescing. It also has a low power electronic system, a compact fluid driving system, and may use direct/unprocessed blood samples and disposable microfluidic cartridges, 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Other objects of the present invention and many of the attendant advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, in which like reference numerals designate like parts throughout the figures thereof and wherein: 
         FIG. 1  is a perspective view of an illustrative portable cytometer in accordance with the present invention; 
         FIG. 2  is a schematic view of the illustrative portable cytometer of  FIG. 1 ; 
         FIG. 3  is a more detailed schematic diagram showing the cytometer of  FIG. 2  with the cover not yet depressed; 
         FIG. 4  is a more detailed schematic diagram showing the cytometer of  FIG. 2  with the cover depressed; 
         FIG. 5  is a schematic diagram showing an illustrative manual fluid driver having a bulb and check valve; 
         FIG. 6  is a graph showing proportional pressure control of an addressable array of microvalves; 
         FIG. 7  is a schematic diagram showing the formation of a flow stream by the hydrodynamic focusing block  88  of  FIG. 3 ; 
         FIG. 8  is a schematic diagram showing an array of light sources and an array of light detectors for analysis of the core stream  160  of  FIG. 7 . 
         FIG. 9  is a graph showing the light intensity produced along the light source axis of  FIG. 8 ; 
         FIG. 10  is a schematic diagram showing an illustrative light source and detector pair of  FIG. 8 ; 
         FIG. 11  is a schematic diagram showing three separate arrays of light sources and detectors, each positioned along a different light source axis that is slightly rotated relative to the central flow axis of the flow stream of  FIG. 7 ; 
         FIG. 12  is a schematic diagram showing an illustrative light source and detector pair of the first array shown in  FIG. 11 ; 
         FIG. 13  is a schematic diagram showing an illustrative light source and detector pair of the second array shown in  FIG. 11 ; 
         FIG. 14  is a schematic diagram showing an illustrative light source and detector pair of the third array shown in  FIG. 11 ; 
         FIG. 15  is a perspective view of an illustrative example of the miniaturized portable cytometer adapted to be worn around the wrist; 
         FIG. 16  is an illustration of a miniaturized portable cytometer cartridge incorporating the scattering and fluorescence optical subsystems; 
         FIG. 17  shows the layout of the scattering and fluorescence detection systems; 
         FIG. 18  is a diagram of the blood hierarchy emphasizing an area of cytometer application; 
         FIGS. 19   a  and  19   b  show the antibody and marker structure associated with the cell or bacteria of interest; 
         FIG. 20  shows the interaction of the light and optics for the fluorescence system; 
         FIGS. 21   a ,  21   b ,  21   c ,  21   d  and  21   e  show the optical structure relative to the flow channel for the scattering and fluorescence systems, respectively; 
         FIG. 22  shows a layout of the scattering and fluorescence detection systems having discrete lenses apart of the flow channel; and 
         FIG. 23  is a perspective view of an illustrative example of the miniaturized portable cytometer having scattering and fluorescence detection systems adapted to be worn around the wrist; 
     
    
    
     DESCRIPTION 
       FIG. 1  is a perspective view of an illustrative miniaturized portable cytometer in accordance with the present invention. The cytometer is generally shown at  10 , and includes a housing  12  and a removable or replaceable cartridge  14 . The illustrative housing  12  includes a base  16 , a cover  18 , and a hinge  20  that attaches the base  16  to the cover  18 . The base  16  includes light sources  22   a  and  22   b , associated optics and the necessary electronics for operation of the cytometer. The cover  12  includes a manual pressurizing element, pressure-chambers with control microvalves, and light detectors  24   a  and  24   b  with associated optics. 
     The removable cartridge  14  preferably receives a sample fluid via a sample collector port  32 . A cap  38  may be used to protect the sample collector port  32  when the removable cartridge  14  is not in use. The removable cartridge  14  preferably performs blood dilution, red cell lysing, and hydrodynamic focusing for core formation. The removable cartridge  14  may be constructed similar to the fluidic circuits available from Micronics Technologies, some of which are fabricated using a laminated structure with etched channels. 
     The removable structure or cartridge  14  is inserted into the housing when the cover  18  is in the open position. The removable cartridge  14  may include holes  26   a  and  26   b  for receiving registration pins  28   a  and  28   b  in the base  16 , which help provide alignment and coupling between the different parts of the instrument. The removable cartridge  14  also preferably includes transparent flow stream windows  30   a  and  30   b , which are in alignment with the arrays of the light sources  22   a  and  22   b , and light detectors  24   a  and  24   b . When the cover is moved to the closed position, and the system is pressurized, the cover  18  provides controlled pressures to pressure receiving ports  34   a ,  34   b , and  34   c  in the removable cartridge  14  via pressure providing ports  36   a,    36   b  and  36   c , respectively. 
     To initiate a test, the cover  18  is lifted and a new cartridge  14  is placed and registered onto the base  16 . A blood sample is introduced into the sample collector  32 . The cover  18  is closed and the system is manually pressurized. Once pressurized, the instrument performs a white blood cell cytometry measurement. The removable cartridge  14  provides blood dilution, red cell lysing, and hydrodynamic focusing for core formation. The light sources  22   a  and  22   b , light detectors  24   a  and  24   b  and associated control and processing electronics perform differentiation and counting of white blood cells based on light scattering fluorescent signals. Rather than using a hinged construction for the housing  12 , it is contemplated that a sliding cartridge slot or any other suitable construction may be used. 
       FIG. 2  is a schematic view of the illustrative cytometer of  FIG. 1 . As above, the base  16  may include light sources  22   a  and  22   b , associated optics and the necessary control and processing electronics  40  for operation of the cytometer. The base  16  may also include a battery  42  for powering the cytometer. The cover  12  is shown having a manual pressurizing element  44 , pressure-chambers  46   a ,  46   b  and  46   c  with control microvalves, and light detectors  24   a  and  24   b  with associated optics. 
     The removable cartridge  14  may receive a sample fluid via the sample collector port  32 . When pressurized by the cover  18 , the removable cartridge  14  performs blood dilution, red cell lysing, and hydrodynamic focusing for core formation in the present device. Once formed, the core is provided down a flow stream path  50 , which passes the flow stream windows  30   a  and  30   b  of  FIG. 1 . The light sources  22   a  and  22   b , and associated optics in the base provide light through and to the core stream via the flow stream windows  30   a  and  30   b . The light detectors  24   a  and  24   b , and associated optics receive scattered and non-scattered light from the core, also via the flow stream windows  30   a  and  30   b,  respectively. The controller or processor  40  receives output signals from the detectors  24   a  and  24   b , and differentiates, identifies and counts selected white blood cells that are present in the core stream. 
     It is contemplated that the removable cartridge  14  may include a fluid control block  48  for helping control the velocity of each of the fluids. In the illustrative example, the fluid control block  48  includes flow sensors for sensing the velocity of the various fluids and reports the velocities to the controller or processor  40 . The controller or processor  40  may then adjust the microvalves associated with pressure-chambers  46   a ,  46   b  and  46   c  to achieve the desired pressures and thus desired fluid velocities for proper operation of the cytometer. 
     Because blood and other biological waste can spread disease, the removable cartridge  14  preferably has a waste reservoir  52  downstream of the flow stream windows  30   a  and  30   b . The waste reservoir  52  receives and stores the fluid of the flow stream in the removable cartridge  14 . When a test is completed, the removable cartridge may be removed and disposed of, preferably in a container compatible with biological waste. 
       FIG. 3  is a more detailed schematic diagram showing the cytometer of  FIG. 2  with the cover  18  not yet depressed.  FIG. 4  is a more detailed schematic diagram showing the cytometer of  FIG. 2  with the cover depressed. The cover  18  is shown having a manual pressurizing element  44 , pressure-chambers  46   a ,  46   b  and  46   c , and control microvalves generally shown at  60 . The light sources and detectors are not shown in these Figures. 
     There are three pressure chambers  46   a ,  46   b  and  46   c,  one for each fluid to be pressurized. In the illustrative example, pressure chamber  46   a  provides pressure to a blood sample reservoir  62 , pressure chamber  46   b  provides pressure to a lyse reservoir  64 , and pressure chamber  46   c  provides pressure to a sheath reservoir  66 . The size and shape of each pressure chamber  46   a ,  46   b  and  46   c  may be tailored to provide the desired pressure characteristics to the corresponding fluid. 
     Pressure chamber  46   a  includes a first pressure chamber  70  and a second pressure chamber  72 . A first valve  74  is provided between the first pressure chamber  70  and the second pressure chamber  72  for controllably releasing the pressure in the first pressure chamber  70  to a second pressure chamber  72 . A second valve  76 , in fluid communication with the second pressure chamber  72 , controllably vents the pressure in the second pressure chamber  72 . Each valve is preferably an array of electrostatically actuated microvalves that are individually addressable and controllable, as described in, for example, co-pending U.S. patent application Ser. No. 09/404,560, entitled “Addressable Valve Arrays for Proportional Pressure or Flow Control”, and incorporated herein by reference. Pressure chambers  46   b  and  46   c  include similar valves to control the pressures applied to the lyse reservoir  64  and sheath reservoir  66 , respectively. Alternatively, each valve may be an array of electrostatically actuated microvalves that are pulse modulated with a controllable duty cycle to achieve a controlled “effective” flow or leak rate. 
     The removable cartridge  14  has pressure receiving ports  34   a ,  34   b , and  34   c  for receiving the controlled pressures from the cover  18 . The controlled pressures are provided to the blood reservoir  62 , lyse reservoir  64  and sheath reservoir  66 , as shown. The lyse reservoir  64  and sheath reservoir  66  are preferably filled before the removable cartridge  14  is shipped for use, while the blood reservoir  62  is filled from sample collector port  32 . A blood sample may be provided to the sample collector port  32 , and through capillary action, the blood sample is sucked into the blood reservoir  62 . Once the blood sample is in the blood reservoir  62 , the cover  18  may be closed and the system may be pressurized. 
     A flow sensor is provided in-line with each fluid prior to hydrodynamic focussing. Each flow sensor  80 ,  100  and  102  measures the velocity of the corresponding fluid. The flow sensors are preferably thermal anemometer type flow sensors, and more preferably microbridge type flow sensor. Microbridge flow sensors are described in, for example, U.S. Pat. Nos. 4,478,076, 4,478,077, 4,501,144, 4,651,564, 4,683,159, and 5,050,429, all of which are incorporated herein by reference. An output signal from each flow sensor  80 ,  100  and  102  is provided to controller or processor  40 . 
     The controller or processor  40  opens the first valve  74  when the velocity of the blood sample drops below a first predetermined value and opens the second valve  76  when the velocity of the blood sample increases above a second predetermined value. Valves  84 ,  86 ,  94  and  96  operate in a similar manner to control the velocities of the lyse and sheath fluids. 
     During operation, and to pressurize the system, the manual pressurizing element  44  is depressed. In the example shown, the manual pressurizing element  44  includes three plungers, with each plunger received within a corresponding one of the first pressure chambers. The plungers create a relatively high non-precision pressure in the first pressure chambers. Lower, controlled pressures are built in the secondary chambers by opening the first valves  70 ,  84  and  94 , which produce a controllable leak into the secondary chambers. If too much pressure builds up in the secondary pressure chambers, the corresponding vent valves  76 ,  86  and  96  are opened to relieve the pressure. 
     When closing the cover  18 , the normally open first valves  74 ,  84  and  94  are closed while the vent valves  76 ,  86  and  96  are open. When a predetermined pressure P is achieved in the first pressure chambers, the vent valves  76 ,  86  and  96  are closed, and the first valves  74 ,  84  and  94  are opened to build a lower pressure P′ in the secondary pressure chambers. The controlled pressure in the secondary pressure chambers provide the necessary pressures to the fluidic circuit of the removable cartridge  14  to produce fluid flow for the blood, lyse and sheath. The velocity of the fluid flow is then measured by the downstream flow sensors  80 ,  100  and  102 . Each flow sensor provides an output signal that is used by the controller or processor  40  to control the operation of the corresponding first valve and vent valve to provide a desired and constant flow rate for each fluid. 
     Downstream valves generally shown at  110  may also be provided. Controller or processor  40  may close downstream valves  110  until the system is pressurized. This may help prevent the blood, lyse and sheath from flowing into the fluid circuit before the circuit is pressurized. In another illustrative example of the invention, downstream valves  110  are opened by mechanical action when the cover is closed. 
       FIG. 5  is a schematic diagram showing an illustrative manual fluid driver having a bulb  100  and check valve  102 . The check valve  102  is preferably a one way valve that allows air in but not out of the first pressure chamber  104 . When the bulb  100  is depressed, the air in the interior  106  of the bulb  100  is forced through the check valve  102  and into the first pressure chamber  104 . Preferably, another one-way vent valve  105  is provided that allows air in from the atmosphere but not out of the interior  106  of the bulb  100 . Thus, when the bulb is released, the one-way vent valve  105  may allow replacement air to flow into bulb  100 . 
     Rather than using a manually operated fluid driver, it is contemplated that any relatively small pressure source may be used including, for example, an electrostatically actuated meso-pump. One such meso-pump is described in, for example, U.S. Pat. No. 5,836,750 to Cabuz, which is incorporated herein by reference. 
       FIG. 6  is a graph showing proportional pressure control produced by an 8×7 addressable array of microvalves. To create the graph shown in  FIG. 6 , 6.5 psi was applied to a first pressure chamber  120 . A small opening was provided to a second pressure chamber  122 . The microvalves are shown at  124 , and vent the pressure in the second pressure chamber  122 . By changing the number of addressable microvalves that are closed, the pressure in the second pressure chamber can be changed and controlled. In the graph shown, the pressure in the second pressure chamber  122  could be changed from about 0.6 psi, when zero of the 8×7 array of microvalves close, to about 6.5 psi, when all of the 8×7 array of microvalves are closed. These low power, micromachined silicon microvalves can be used for controlling pressures up to 10 psi and beyond. 
       FIG. 7  is a schematic diagram showing the formation of a flow stream and core by the hydrodynamic focusing block  88  of  FIG. 3 . The hydrodynamic focusing block  88  receives blood, lyse and sheath at controlled velocities from the fluid driver. The blood is mixed with the lyse, causing the red blood cells to be removed. The lysing solution may have a pH lower than that of the red blood cells. This is often referred to as red cell lysing or lyse-on-the-fly. The remaining white blood cells are provided down a central lumen  150 , which is surrounded by sheath fluid to produce a flow stream  50 . The flow stream  50  includes a core stream  160  surrounded by the sheath fluid  152 . The dimensions of the channel are reduced as shown so that the white blood cells  154  and  156  are in single file. The velocity of the sheath fluid is preferably about 9 times that of the core stream  160 . However, the velocity of the sheath fluid and core stream  160  remain sufficiently low to maintain laminar flow in the flow channel. 
     Light emitters  22   a  and  22   b , and associated optics are preferably provided adjacent one side of the flow stream  50 . Light detectors  24   a  and  24   b , and associated optics are provided on another side of the flow stream  50  for receiving the light from the light emitters  22   a  and light from fluorescing particles via the flow stream  50 . The output signals from the light detectors  24   a  and  24   b  are provided to controller or processor  40 , wherein they are analyzed to identify and/or count selected white blood cells in the core stream  160 . 
       FIG. 8  is a schematic diagram showing an array  22   a  of light sources and an array  24   b  of light detectors for analysis of the core stream  160  via scattering of  FIG. 7 . The light sources are shown as “+” signs and the detectors are shown at boxes. In the example shown, the array of light sources is provided adjacent one side of the flow stream  50 , and the array of light detectors is provided adjacent the opposite side of the flow stream. Each of the light detectors is preferably aligned with a corresponding one of the light sources. The array of light sources and the array of light detectors are shown arranged along a light source axis  200  that is slightly rotated relative to the axis  202  of the flow stream  50 . 
     The array  22   a  of light sources is preferably an array of lasers such as vertical cavity surface emitting lasers (VCSELs) fabricated on a common substrate. Because of their vertical emission, VCSELs are ideally suited for packaging in compact instruments such as a miniaturized portable cytometer. Such cytometer may be wearable on a person&#39;s body. Preferably, the VCSELs are “red” VCSELs that operate at wavelengths that are less than the conventional 850 nm, and more preferably in the 670 nm to 780 nm range. Red VCSELs may have a wavelength, power and polarization characteristic that is ideally suited for scatter measurements. 
     Some prior art cytometer bench models use a single 9 mW edge-emitting laser with a wavelength of 650 nm. The beam is focussed to a 10×100 micron elongated shape to cover the uncertainty in particle position due to misalignment and width of the core stream. In contrast, the output power of the red VCSELs of the present invention, operating at 670 nm, is typically around 1 mW for a 10×10 micron emitter and 100-micron spacing. Thus, the total intensity of the light from a linear array of ten red VCSELs may be essentially the same as that of some prior art bench models. 
     Using a linear array of lasers oriented at an angle with respect to the flow axis  202  offers a number of important advantages over the single light source configuration of the prior art. For example, a linear array of lasers may be used to determining the lateral alignment of the path of the particles in the core steam. One source of uncertainty in the alignment of the particle stream is the width of the core flow, which leads to statistical fluctuations in the particle path position. These fluctuations can be determined from analysis of the detector data and can be used by the controller or processor  40  to adjust the valves of the fluid driver in order to change the relative pressures that are applied to the sample fluid and the supporting fluids to change the alignment of the selected particles in the flow stream. 
     To determine the lateral alignment of the cells in the fluid stream  50 , the cells pass through several focussed spots produced by the linear array of VCSELs. The cells produce a drop in signal in the corresponding in-line reference detectors. The relative strengths of the signals are used by the controller or processor  40  to determine the center of the particle path and a measure of the particle width. 
     For determining particle path and size, the lasers  22   a  are preferably focussed to a series of Gaussian spots  214  (intensity on the order of 1000 W/cm 2 ) in the plane of the core flow. The spots  214  are preferably about the same size as a white blood cell (10-12 um). Illustrative Gaussian spots  214  are shown in  FIG. 9 . Arrays  24   a  of detectors and their focussing optics are provided on the opposite side of the fluid stream  50 . Lenses with fairly large F-numbers are used to provide a working space of several hundred microns for the cytometer section of the removable cartridge. 
     Another advantage of using a linear array  22   a  of lasers rather than a single laser configuration is that the velocity of each cell may be determined. Particle velocity can be an important parameter in estimating the particle size from light scatter signals. In conventional cytometry, the particle velocity is extrapolated from the pump flow rates. A limitation of this approach is that the pumps must be very precise, the tolerance of the cytometer flow chambers must be tightly controlled, no fluid failures such as leaks can occur, and no obstructions such as microbubbles can be introduced to disturb the flow or core formation. 
     To determine the velocity of each cell, the system may measure the time required for each cell to pass between two adjacent or successive spots. For example, and with reference to  FIG. 8 , a cell may pass detector  208  and then detector  210 . By measuring the time required for the cell to travel from detector  208  to detector  210 , and by knowing the distance from detector  208  to detector  210 , the controller or processor  40  can calculate the velocity of the cell. This would be an approximate velocity measurement. This is often referred to as a time-of-flight measurement. Once the velocity is known, the time of travel through the spot on which the particle is centered (a few microseconds) may provide a measure of particle length and size. 
     It is contemplated that the particle velocity can also be used to help control the fluid driver. To reduce the size, cost and complexity of the present invention, the replaceable cartridge of  FIG. 1  may be manufactured from a plastic laminate or molded parts. While such manufacturing techniques may provide inexpensive parts, they are typically less dimensionally precise and repeatable, with asymmetrical dimensions and wider tolerance cross-sections. These wider tolerances may produce variations in particle velocity, particularly from cartridge to cartridge. To help compensate for these wider tolerances, the time-of-flight measurement discussed above can be used by the controller or processor  40  to adjust the controlled pressures applied to the blood, lyse and sheath fluid streams such that the particles in the core stream have a relatively constant velocity. 
     To further evaluate the cell size, it is contemplated that laser beams may be focused both along the cell path and across the cell path. Additionally, multiple samples across the cell may be analyzed for texture features, to correlate morphological features to other cell types. This may provide multiple parameters about cell size that may help separate cell types from one another. 
     Another advantage of using a linear array  22   a  of lasers rather than a single layer configuration is that a relatively constant light illumination may be provided across the flow channel. This is accomplished by overlapping the Gaussian beams  214  from adjacent VCSELs  22   a , as shown in  FIG. 9 . In prior art single laser systems, the light illumination across the flow channel typically varies across the channel. Thus, if a particle is not in the center of the flow channel, the accuracy of subsequent measurements may be diminished. 
     To perform the above described measurements, each detector  24   a  in  FIG. 8  may be a single in-line detector. To measure FALS and SALS scatter, however, each detector  24   a  may further include two annular detectors disposed around the in-line detector, as shown in  FIG. 10 . Referring to  FIG. 10 , a VCSEL  218  is shown providing light in an upward direction. The light is provided through a lens  220 , which focuses the light to a Gaussian spot in the plane of the core flow. Lens  220  may be a microlens or the like, which is either separate from or integrated with the VCSEL  218 . The light passes through the core flow, and is received by another lens  222 , such as a diffractive optical element. Lens  222  provides the light to in-line detector  226  and annular detectors  228  and  230 . The in-line detector  226  detects the light that is not significantly scattered by the particles in the core stream. Annular detector  228  detects the forward scatter (FALS) light, and annular detector  230  detects the small angle scatter (SALS) light. 
       FIG. 11  shows another illustrative example of the present invention that includes three separate arrays of light sources and light detectors. Each array of light sources and light detectors are positioned along a different light source axis that is slightly rotated relative to the central flow axis of the flow stream. By using three arrays, the optics associated with each array may be optimized for a particular application or function. For detecting small angle scattering (SALS), laser light that is well-focussed on the plane of the core flow is desirable. For detecting forward scattering (FALS), collimated light is desirable. 
     Referring specifically to  FIG. 11 , a first array of light sources and light detectors is shown at  300 . The light sources and light detectors are arranged in a linear array along a first light source axis. The first light source axis is rotated relative to the flow axis of the flow stream. The light sources and light detectors may be similar to that described above with respect to  FIG. 8 , and preferably are used to measure, for example, the lateral alignment of the cells in the flow stream, the particle size, and the velocity of the particles. 
       FIG. 12  is a schematic diagram showing an illustrative light source and detector pair of the first array  300  shown in  FIG. 11 . A VCSEL  302  is shown providing light in an upward direction. The light is provided through a lens  304 , which focuses the light to a Gaussian spot in the plane of the core flow. The light passes through the core flow, and is received by another lens  306 . Lens  306  provides the light to in-line detector  308 . The in-line detector  308  detects the light that is not significantly scattered by the particles in the core stream. 
     A second array of light sources and light detectors is shown at  310 . The light sources are arranged in a linear array along a second light source axis that is rotated relative to the flow axis of the flow stream. The light detectors include three linear arrays of light detectors. One array of light detectors is positioned in line with the linear array of light sources. The other two linear arrays of light detectors are placed on either side of the in-line array of light detectors, and are used for measuring the small angle scattering (SALS) produced by selected particles in the flow stream. 
       FIG. 13  is a schematic diagram showing an illustrative light source and corresponding detectors of the second array shown in  FIG. 11 . A VCSEL  320  is shown providing light in an upward direction. The light is provided through a lens  322 , which focuses the light to a Gaussian spot in the plane of the core flow. The light passes through the core flow, and is received by another lens  324 , such as a diffractive optical element (DOE)  324 . Lens  324  provides the light to the in-line detector  326  and the two corresponding light detectors  328  and  330  placed on either side of the in-line light detector  326 . 
     The in-line detector  326  may be used to detect the light that is not significantly scattered by the particles in the core stream. Thus, the in-line linear array of light detectors of the second array  302  may be used to provide the same measurements as the in-line array of detectors of the first array  300 . The measurements of both in-line arrays of detectors may be compared or combined to provide a more accurate result. Alternatively, or in addition, the in-line detectors of the second array  302  may be used as a redundant set of detectors to improve the reliability of the cytometer. 
     It is contemplated that the in-line detectors of the second array  302  may also be used in conjunction with the in-line detectors of the first array  300  to more accurately determine the time-of-flight or velocity of the particles in the flow stream. The measurement may be more accurate because the distance between detectors may be greater. As indicated above, by knowing the velocity of the particles, small variations in the flow rate caused by the fluid driver can be minimized or removed by the controller. 
     Light detectors  328  and  330  of  FIG. 13  are used to measure the small angle scattering (SALS) produced by selected particles in the flow stream. The light detectors  328  and  330  are therefore preferably spaced sufficiently from the in-line detector  326  to intercept the small angle scattering (SALS) produced by selected particles in the flow stream. 
     Referring back to  FIG. 11 , a third array of light sources and light detectors  350  is preferably provided to measure the forward angle scattering (FALS) produced by selected particles in the flow stream. The light sources are arranged in a linear array along a third light source axis that is rotated relative to the flow axis of the flow stream. Each light source preferably has a corresponding light detector, and each light detector is preferably annular shaped with a non-sensitive region or a separate in-line detector in the middle. The annular shaped light detectors are preferably sized to intercept and detect the forward angle scattering (FALS) produced by selected particles in the flow stream. 
       FIG. 14  is a schematic diagram showing an illustrative light source and detector pair of the third array of light sources and light detectors  350  shown in  FIG. 11 . A VCSEL  360  is shown providing light in an upward direction. The light is provided through a lens  362  such as a collimating lens, which provides substantially collimated light to the core flow. As indicated above, collimated light is desirable for detecting forward scattering (FALS) light. The light passes through the core flow, and is received by another lens  364 . Lens  364  provides the received light to the annular shaped detector  368 . 
     The annular shaped detector  368  is preferably sized to intercept and detect the forward angle scattering (FALS) produced by selected particles in the flow stream. A non-sensitive region or a separate in-line detector  370  may be provided in the middle of the annular shaped detector  368 . If a separate in-line detector  370  is provided, it can be used to provide the same measurement as the in-line detectors of the first array  300  and/or second array  302 . When so provided, the measurements from all three in-line arrays of detectors of first array  300 , second array  302  and third array  350  may be compared or combined to provide an even more accurate result. The in-line detectors of the third array  302  may also be used as another level or redundancy to improve the reliability of the cytometer. 
     It is contemplated that the in-line detectors of the third array  350  may also be used in conjunction with the in-line detectors if the first array  300  and/or second array  302  to more accurately determine the time-of-flight or velocity of the particles in the flow stream. The measurement may be more accurate because the distance between detectors may be greater. As indicated above, by knowing the velocity of the particles, small variations in the flow rate caused by the fluid driver can be minimized or removed by the controller. 
     By using three separate arrays of light sources and detectors, the optics associated with each array can be optimized for the desired application. As can be seen, the optics associated with the first array  300  are designed to provide well-focussed laser light on the plane of the core flow. This helps provide resolution to the alignment, size and particle velocity measurements performed by the first array  300 . Likewise, the optics associated with the second array  302  are designed to provide well-focussed laser light on the plane of the core flow. Well focussed light is desirable when measuring the small angle scattering (SALS) produced by selected particles in the flow stream. Finally, the optics associated with the third array  350  are designed to provide collimated light to the core flow. As indicated above, collimated light is desirable when measuring forward angle scattering (FALS) produced by selected particles in the flow stream. 
       FIG. 15  is a perspective view of an illustrative example of the miniaturized portable cytometer of the present invention adapted to be worn around the wrist. This cytometer  400  may be similar to that shown in  FIG. 1 . A band  402  secures cytometer  400  to the wrist of a user. 
     As indicated above, the user may obtain a removable cartridge and provide a blood sample to the sample collector port  32  (see  FIG. 1 ) of the removable cartridge. The blood sample may be collected by, for example, a finger prick. The user may then insert the removable cartridge into the housing, and manually pressurize the system. The miniaturized portable cytometer may then provide a reading that indicates if the user should seek medical treatment. The reading may be a visual reading, an audible sound or any other suitable indicator. 
     Rather than obtaining the blood sample by a finger prick or the like, it is contemplated that a catheter  404  or the like may be inserted into a vein of the user and attached to the sample collector port  32 . This may allow the system to automatically collect a blood sample from the user whenever a reading is desired. Alternatively, it is contemplated that the miniaturized portable cytometer may be implanted in the user, with the sample collector port  32  connected to a suitable blood supply. 
       FIG. 16  shows a cytometer cartridge  500  showing a scattering optical subsystem  501  and fluorescent optical subsystem  502 . Optical subsystem  501  includes windows or openings  30   a  on each side of flow channel  530  and optical subsystem  502  includes windows or openings  30   b . In each subsystem, there is a window or opening on each side of flow channel  530 . The openings may have optical inserts or lenses. This cytometer may be implemented for wearing, attachment on, or insertion in a person&#39;s body 
       FIG. 17  shows systems  503  and  504  which incorporate optical subsystems  501  and  502 , respectively. System  503  also includes VCSEL array  22   a  and detector array  24   a for scattering measurements of particles, such as white blood cells, in core stream  160 . This system may be used for the counting and classification of lymphocytes and neutrophils. Self-alignment is may be enabled by a red VCSEL-array based optical subsystem. Illustrative examples of scattering system  503  are described above. 
     System  504  is a fluorescent exciting and detection mechanism which may be used for identifying and counting specific subclasses of white blood cells and blood-based proteins. The detection of subclasses of white blood cells is enabled by the availability of suitable antibodies, many of which are commercially available in a fluorescently conjugated form.  FIG. 18  shows an outline sketch of blood composition and the cells that may be subject to counting and identification by fluorescent system  504 . The red blood cells are removed from the sample to be looked with the cytometer by lysing as noted above. The platelets are kept as the small size does not affect the results of the cytometer when checking the white blood cells. For an illustrative example, the CD4-positive T-cells  505 , shown in the structure of  FIG. 18  have proportions and counts in blood tbat may be very important in following a clinical course of an HIV infection. An antibody with a marker added that associates with CD4 may be mixed in the sample of blood to get a resultant “Y”-looking structure of the antibody (AB)  506  and its marker (M)  507 , attached to CD4 cell  505 , as shown in  FIG. 19   a . Light source  22   b  may emit light which may be absorbed by marker  507 . In response, marker  507  fluoresces and emits light of a particular wavelength which may be detected to identify CD4 cell  505 . 
     Checking blood for anthrax may be another application of the present cytometer. Antibodies  508  for the anthrax-causing bacteria  509  may be mixed with the blood sample. The antibodies may associate with bacteria  509 . The antibodies may have markers  510  that fluoresce upon impingement of light. The “Y” structure of antibody  508  is shown in  FIG. 19   b . Markers  510  emit a light of a particular bandwidth which may be different from the bandwidth of marker  507  of antibody  506  for CD 4  cell  505 . So the anthrax problem may be identified separately from the HIV problem in the same blood sample test by the fluorescent emissions having different wavelengths, colors or signatures. The number of different problems being detected at the same time in the same blood sample may be many more than two. 
     For another illustrative example, Neupogen R  (a type of protein) has been regarded as used to provide neutrophil counts in cancer patients undergoing myelosuppressive chemotherapy. While doing this therapy, there may a need to accurately monitor the white blood cell counts (specifically neutrophils, monocytes and platelet counts during the Neupogen R  therapy period). The present cytometer may be used by untrained personnel to monitor such chemotherapy patients in their homes. 
     The present miniaturized portable cytometer may be use in biowarfare. It may be used for quantitative detection and identification of biowarfare agents. This detection and identification may be based antibody-antigen type immunoassay that may be implemented with fluorescent measurements. The environment, water and food may be monitored for any possible presence of biological agents. It would involve sample collection and preparation appropriated for the present cytometer. Other applications of the cytometer may include high throughput analysis (using the fluorescent detection features) and sequencing of DNA and RNA, studying the response of cell to potential drugs, immunophenotyping of leukemia and lymphomas and monitoring residual disease in cancer patients, and cell sorting and cell isolation, including high-speed separation of rare event populations. Note that the above-mentioned applications, and other applications and uses may be accomplished with the single, portable, miniaturized, integrated scattering and multi-color fluorescent, low-power, low-cost cytometry instrument having a compact precision fluid driving system, not requiring operator intervention or adjustment during the analytical phase, not requiring trained personnel to operate the instrument, and using sanitary, disposable plastic- or other material-based microfluidic cartridges  14  having integrated optics and internal blood sample processing, among other features. 
     System  504  of  FIG. 17  has a laser light source  22   b  positioned to direct light  511  at particles  512  flowing single file through flow channel  530 . For illustrative purposes, particles  512  may include structures  513  and  514  of  FIGS. 19   a  and  19   b , respectively. Light  511  may be from a red or a blue laser source, such as a light emitting diode (LED), which may have a bandwidth of, for example, 650 to 700 nanometers or 380 to 420 nanometers, respectively. Other types of sources having appropriate wavelengths may be used for light  511 . As light  511  impinges fluorescent markers  507  and  510 , these markers fluoresce and emit light  515  and  516 , respectively. Since the markers are different from each other, light  515  and light  516  have different wavelengths. Thus, structures  513  and  514  not only may be identifiable by the wavelengths of their emitted light but can be differentiated from each other in the same sample, blood or otherwise. Light  515  and  516  may go to a dichroic beam splitter  517  which separates the two beams by directing each of them in different directions. Beam  516  may go to a fluorescence photo detector  518  for detection and conversion of light  516  into an electrical signal  520  to processor  40 . Beam  515  may go to a fluorescence photo detector  521  for detection and conversion of light  515  into an electrical signal  522  to processor  40 . Band pass filter  519 , which is in the path of beam  516 , may filter out light  511  from light source  22   b  that managed to be present in beam  516 . Band pass filter  523  may serve the same purpose for beam  515  as filter  519  for beam  515 . A mirror  524  may be used to redirect beam  515  for purposes of detector  521  location for the possibility of more compact packaging of detection system  504  or for other reasons. Mirror  524  may on the other hand be another dichroic beam splitter for splitting out light  525  of a wavelength different from that of beams  515  and  516 . More splitters might be used in a cascade-like or other structure to split out light of still other frequencies. Also going to processor  40  is a signal from detector array  24   a  of scattering detection system  503 . 
     Splitter  517  may be replaced with other mechanisms for separating out the light of various wavelengths or selecting certain wavelengths. They may include notch and step function filters of various kinds, tunable diffraction gratings, thin film dielectric stacks, mirror beam splitters, photonic bandgap filters, photonic crystals, tunable band pass filters, etalon comb and other structures, wafers having light guides with structural or other filtering, silicon or glass wafers having a waveguide and perforations of a specific size and pitch for absorbing/filtering, and so on. 
       FIG. 20  shows the structure of the fluorescence optical subsystem  502 . A beam  511  may be emitted by light source  22   b  and focused onto a particle  512  by a microlens  526  through window  30   b . Light beam  511  may or may not be collimated. Particle  512  may have a marker that fluoresces and emits a light beam  515 ,  516  through window  30   b , a thin film coating filter  527  and a microlens  528 , respectively. Filter  527  may filter out light  511  from light source  22   b . Filter  527  may be a dielectric stack situated under lens  528  and be a notch or step function filter to block source  22   b  light  511 . Lens  528  may focus fluorescent light emitted from the marker into a beam  515 / 516  which may go on to a beam splitter such as splitter  517 . Beam  515 / 516  may or may not be collimated. An opaque or absorptive layer  529  is formed around or before and after window  30   b  or lens  528  on a glass, quartz or plastic (laminated or not) substrate  531  of flow channel  530 . Layer  529  may block any light  511  emanating from light source  22   b  from exiting out with fluorescent light  515 / 516 . Layer or blocking filter  529  may a thin film that is black or opaque to the wavebands desired to be blocked. Filter  529  could be a notch or step function filter. The other glass, quartz or plastic (laminated or not) substrate  532  forms flow channel  530  for the core flow of particles  512 . The material of substrates  531  and  532 , windows  30   b  and lens  526  and  528  should not contain ingredients that may fluoresce. In one illustrative example, the direction of light  511  from source  22   b  may be about 90 degrees relative to the direction of fluorescent light  515 / 516  emitted from particle  512 . This angle between source light  511  and emitted fluorescent light  515 / 516  may effectively reduce or eliminate source light emanating out with fluorescent light  515 / 516 . The angle of the direction of light  511  from source  22   b  in the example may be about 45 degrees relative to the direction of the longitudinal dimension flow channel  530  or the direction of the core flow of particles  512 . However, in some applications, the angle between the directions of light  511  and light  515 / 516  may be between 0 and 120 degrees. 
       FIG. 21   a  shows an end view of flow channel  530  for scattering optical subsystem  501  and  FIG. 21   b  shows an end view of flow channel  530  for fluorescence optical subsystem  502 . The thicknesses of substrates  531  and  532  are about 100 to 200 microns. The thicknesses of windows  30   a  and  30   b  are about 25 to 30 microns. Microlenses  526  and  528  may be diffractive or refractive, plastic or glass and be aspheric lenses about 500 microns in diameter. Channel  533  may be laser cut. 
       FIGS. 21   c ,  21   d  and  21   e  are variations of  FIGS. 21   a  and  21   b .  FIG. 21   c  shows a flow channel having windows or openings  30   a  and  30   b . Openings or windows  30   a  and  30   b  may be one window on each side of the flow channel, respectively. The openings may have optical inserts or lenses in them. Micro lenses  526  and  528  or other types of lenses may be formed on, attached to, inserted in, situated on or formed as an integral portion of openings or windows  30   b  and  30   a  which may be one piece on each side of the channel.  FIG. 21   d  illustrates windows  30   a  and  30   b  without micro lenses attached or formed on them, but with lenses  541  and  542  situated at a certain and appropriate distance from them.  FIG. 21   e  shows a window configuration with both micro lenses  526  and  528  along with detached lenses  541  and  542 , respectively. 
       FIG. 22  shows a diagram of cytometer configuration of  FIG. 17 , but with the placement of lenses  541  and  542 . As noted above, windows and openings  30   b  may or may not have micro lenses in addition to lenses  541  and  542 . 
       FIG. 23  is a perspective view of an illustrative example of a miniaturized portable cytometer having both scattering and fluorescent detection and monitoring adapted to be worn around the wrist or palm. This cytometer  600  may be similar to that shown in  FIGS. 1 and 16 . A band  602  secures the miniaturized portable cytometer  600  to the wrist of a user. 
     As indicated above, the user may obtain a removable cartridge and provide a blood sample to the sample collector port  32  (see  FIGS. 1 ,  16 ,  17  and  22 ) of the removable cartridge. The blood sample may be collected by, for example, a finger prick. The user may then insert the removable cartridge into the housing, and manually pressurize the system. The miniaturized and portable cytometer may then provide a reading that indicates if the user should seek medical treatment. The reading may be a visual reading, an audible sound or any other suitable indicator. 
     Rather than obtaining the blood sample by a finger prick or the like, it is contemplated that a catheter  604  or the like may be inserted into a vein of the user and attached to sample collector port  32 . This may allow the system to automatically collect a blood sample from the user whenever a reading is desired. Alternatively, it is contemplated that the miniaturized portable cytometer may be implanted in the user, with sample collector port  32  connected to a suitable blood supply. 
     Although the invention has been described with respect to at least one illustrative example, many variations and modifications will become apparent to those skilled in the art upon reading the present specification. It is therefore the intention that the appended claims be interpreted as broadly as possible in view of the prior art to include all such variations and modifications.