Patent Abstract:
An apparatus having a disposable cytometer cartridge containing pumps, pressure chambers, reservoirs, flow sensors, flow channels, and microfluidic circuits with fluid operations on the cartridge. The circuits may include mesopumps and mesovalves embedded in the chip, card or cartridge. The apparatus may have multiple detecting, analyzing and identification capabilities of blood or other fluids of interest. The sample to be tested may be entered in the disposable microfluidic cartridge which in turn is insertable in a hand-holdable or portable cytometer instrument. This apparatus may have significant application in biological warfare agent detection, water analyses, environmental checks, hematology, and other clinical and research fields.

Full Description:
[0001]    This present application is a continuation-in-part of U.S. patent application No. 10/304,773, filed Nov. 26, 2002, which 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 B1, and claims the benefit thereof. Also, this present application is a continuation-in-part of U.S. patent application Ser. No. 10/980,685, filed Nov. 3, 2004, which is a division of U.S. patent application Ser. No. 10/174,851, filed Jun. 19, 2002, now U.S. Pat. No. 6,837,476, and claims the benefit thereof. Also, this present application is a continuation-in-part of U.S. patent application Ser. No. 10/340,231, filed Jan. 10, 2003, which is a division of U.S. patent application Ser. No. 09/586,093, filed Jun. 2, 2000, now U.S. Pat. No. 6,568,286 B1, and claims the benefit thereof. All of the above-mentioned patent documents are incorporated herein by reference. 
         [0002]    The present invention is related to U.S. patent application Ser. No. 10/905,995, filed Jan. 28, 2005, by Cabuz et al., entitled “Mesovalve Modulator”, and incorporated herein by reference. Also, the present invention is related U.S. patent application Ser. No. 11/018,799, filed Dec. 21, 2004, by Cabuz et al., entitled “Media Isolated Electrostatically Actuated Valve”, and incorporated herein by reference. These applications are owned by the same entity that owns the present invention. 
         [0003]    The present invention is also related to U.S. Pat. No. 6,549,275 B1, issued Apr. 15, 2003 to Cabuz et al., and entitled “Optical Detection System for Flow Cytometry”; U.S. Pat. No. 6,382,228 B1, issued May 7, 2002 to Cabuz et al., and entitled “Fluid Driving System for Flow Cytometry”; U.S. Pat. No. 6,700,130 B2, issued Mar. 2, 2004 to Fritz, and entitled “Optical Detection System for Flow Cytometry”; U.S. Pat. No. 6,729,856 B2, issued May 4, 2004, to Cabuz et al., and entitled “Electrostatically Actuated Pump with Elastic Restoring Forces”; U.S. Pat. No. 6,255,758 B1, issued Jul. 3, 2001, to Cabuz et al., and entitled “Polymer Microactuator Array with Macroscopic Force and Displacement”; U.S. Pat. No. 6,240,944 B1, issued June 5, 2001 to Ohnstein et al., and entitled “Addressable Valve Arrays for Proportional Pressure or Flow Control”; U.S. Pat. No. 6,179,586 B1, issued Jan. 30, 2001 to Herb et al., and entitled “Dual Diaphragm, Single Chamber Mesopump”; and U.S. Pat. No. 5,836,750, issued Nov. 17, 1998 to Cabuz, and entitled “Electrostatically Actuated Mesopump Having a Plurality of Elementary Cells”; all of which are incorporated herein by reference. These patents are owned by the same entity that owns the present invention. 
     
    
     BACKGROUND 
       [0004]    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. 
         [0005]    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 may be 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. 
         [0006]    Flow cytometry is currently used in a wide variety of applications including hematology, immunology, genetics, food science, pharmacology, microbiology, parasitology, oncology, biological agent detection, and environmental science, 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 
       [0007]    The present invention can overcome many of the disadvantages of the related art by providing a highly miniaturized portable and wearable apparatus (e.g., cytometer) that is usable at remote locations, such as at home or in the field. The apparatus may incorporate fluid devices and operations on a disposable cartridge, chip or card, with optical and electrical interfaces external to the cartridge. Such an apparatus 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. The apparatus may also be used in non-medical applications such as those in various environmental and industrial areas. 
         [0008]    In military applications, the apparatus may be a 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. 
         [0009]    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. 
         [0010]    The first clinical symptoms of infection from some bacterial agents such as bacillus anthrax appear after one to six days. In 99 percent 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. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
         [0011]      FIG. 1  is a perspective view of an illustrative portable cytometer in accordance with the present invention; 
           [0012]      FIG. 2  is a schematic view of the illustrative portable cytometer of  FIG. 1 ; 
           [0013]      FIG. 3  is a more detailed schematic diagram showing the cytometer of  FIG. 2  with the cover not yet depressed; 
           [0014]      FIG. 4  is a more detailed schematic diagram showing the cytometer of  FIG. 2  with the cover depressed; 
           [0015]      FIG. 5  is a schematic diagram showing an illustrative manual fluid driver having a bulb and check valve; 
           [0016]      FIG. 6  is a graph showing proportional pressure control of an addressable array of microvalves; 
           [0017]      FIG. 7  is a schematic diagram showing the formation of a flow stream by a flow mechanism block  88  of  FIG. 3 ; 
           [0018]      FIG. 8  is a schematic diagram showing an array of light sources and an array of light detectors for analysis of the core stream of  FIG. 7 ; 
           [0019]    FIG. 9 is a graph showing the light intensity produced along the light source axis of  FIG. 8 ; 
           [0020]      FIG. 10  is a schematic diagram showing an illustrative light source and detector pair of  FIG. 8 ; 
           [0021]      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 ; 
           [0022]      FIG. 11   a  is a three dimensional illustration of an array of light sources and an array of light detectors positioned along a light source and detector axis that is not parallel to the central flow axis of the flow stream; 
           [0023]      FIG. 12  is a schematic diagram showing an illustrative light source and detector pair of the first array shown in  FIG. 11 ; 
           [0024]      FIG. 13  is a schematic diagram showing an illustrative light source and detector pair of the second array shown in  FIG. 11 ; 
           [0025]      FIG. 14  is a schematic diagram showing an illustrative light source and detector pair of the third array shown in  FIG. 11 ; 
           [0026]      FIG. 15  is a perspective view of an illustrative example of the miniaturized portable cytometer adapted to be worn around the wrist; 
           [0027]      FIG. 16  reveals a disposable cytometer cartridge containing the pumps, pressure chambers, reservoirs, flow sensors, and a flow mechanism with a flow channel on the cartridge; 
           [0028]      FIG. 17   a  is another version of the cartridge where the liquid devices and operations occur on cartridge; 
           [0029]      FIG. 17   b  is like the version of  FIG. 17   a  except that the processor is situated in the cartridge; 
           [0030]      FIGS. 18   a - 18   d  show a various stages of fluid flow in a microfluidic circuit in cartridge or chip; and 
           [0031]      FIGS. 19   a  and  19   b  reveal an application in a microfluidic circuit of mesopumps and mesovalves embedded in a chip, card or cartridge  14 ; 
           [0032]      FIGS. 20   a  and  20   b  reveal an illustrative example of a mesovalve in a closed state and an open state, respectively; 
           [0033]      FIG. 21  is a diagram of a the cartridge having a mesovalve with open loop control; 
           [0034]      FIG. 22  is a diagram of a fluid pump on a cartridge with open loop control; 
           [0035]      FIG. 23  shows a liquid pump and flow sensor on a cartridge, with closed loop control; 
           [0036]      FIG. 24  shows a gas pump and flow sensor on a cartridge, with closed loop control; 
           [0037]      FIG. 25  shows a gas pump, buffer and liquid reservoir on a cartridge, with open loop control; 
           [0038]      FIG. 26  is similar to  FIG. 25 , except the components are off the cartridge; 
           [0039]      FIG. 27  is similar to  FIG. 25 , except it also includes a flow sensor and closed loop control; 
           [0040]      FIG. 28  is similar to  FIG. 27 , except the components are off the cartridge; 
           [0041]      FIG. 29  is similar to  FIG. 27 , except it has the flow sensor off the cartridge and has a pressure chamber; 
           [0042]      FIG. 30  is similar to  FIG. 29 , except the flow sensor is on the cartridge; 
           [0043]      FIG. 31  is similar to  FIG. 30 , except the pressure chamber has a different configuration; and 
           [0044]      FIG. 32  is similar to  FIG. 31 , except the components are shown as off the cartridge. 
           [0045]      FIG. 33  is similar to  FIG. 30 , except the components are shown off the cartridge. 
       
    
    
     DESCRIPTION 
       [0046]    In an illustrative example of the present invention, a portable miniaturized cytometer may be 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. 
         [0047]    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. 
         [0048]    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. 
         [0049]    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. 
         [0050]    The controlled sample fluid and supporting fluids are provided to a fluidic circuit. The fluidic circuit may perform 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. 
         [0051]    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 (or “card” as it may sometimes be referred to) that is inserted into the housing. The fluidic circuit may dilute the blood sample, perform red cell lysing, and perform hydrodynamic focusing for flow and core stream formation. The light sources may be 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. 
         [0052]    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. 
         [0053]    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. 
         [0054]    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. 
         [0055]    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. 
         [0056]    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. 
         [0057]    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. 
         [0058]    This miniaturized portable cytometer may have 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, 
         [0059]      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. 
         [0060]    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  may perform 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. 
         [0061]    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. 
         [0062]    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  may provide 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 may 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. 
         [0063]      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. 
         [0064]    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  may perform blood dilution, red cell lysing, and hydrodynamic focusing for core formation in the present device. Once formed, the core may be 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. 
         [0065]    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. 
         [0066]    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. 
         [0067]      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. 
         [0068]    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. 
         [0069]    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. 
         [0070]    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. 
         [0071]    A flow sensor may be provided in-line with each fluid prior to hydrodynamic focusing. Each flow sensor  80 ,  100  and  102  may measure the velocity of the corresponding fluid. The flow sensors may be thermal anemometer type flow sensors and/or microbridge type flow sensor. Microbridge flow sensors are described in, for example, U.S. Pat. No. 4,478,076, U.S. Pat. No. 4,478,077, U.S. Pat. No. 4,501,144, U.S. Pat. No. 4,651,564, U.S. Pat. No. 4,683,159, and U.S. Pat. No. 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 . 
         [0072]    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 Page  15  of  78  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. 
         [0073]    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. 
         [0074]    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. 
         [0075]    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. 
         [0076]      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 . 
         [0077]    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 mesopump. One such mesopump is described in, for example, U.S. Pat. No. 5,836,750 to Cabuz, which is incorporated herein by reference. 
         [0078]      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. 
         [0079]      FIG. 7  is a schematic diagram showing the formation of a flow stream and core by a flow mechanism block  88 , which may provide hydrodynamic focusing, of  FIG. 3 . The block  88  may receive blood, lyse and sheath at controlled velocities from the fluid driver. The blood may be 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. 
         [0080]    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 . 
         [0081]      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 . 
         [0082]    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. 
         [0083]    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. 
         [0084]    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. 
         [0085]    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. 
         [0086]    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 focusing 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. 
         [0087]    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. 
         [0088]    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. 
         [0089]    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. 
         [0090]    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. 
         [0091]    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. 
         [0092]    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. 
         [0093]      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. 
         [0094]    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. 
         [0095]      FIG. 11   a  is a three dimensional illustration of an array of light sources  351  and an array of light detectors  353  positioned along a light source axis  355  and detector axis  357 , respectively, which are not parallel (i.e., are statically rotated) relative to the central flow axis of the flow stream  359 . Axes  355 ,  357  and  361  are typically parallel to one another. Line  361  is an axis of light spots across flow stream  359 . 
         [0096]      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. 
         [0097]    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. 
         [0098]      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 . 
         [0099]    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. 
         [0100]    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. 
         [0101]    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. 
         [0102]    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. 
         [0103]      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 . 
         [0104]    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. 
         [0105]    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. 
         [0106]    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. 
         [0107]      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. 
         [0108]    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. 
         [0109]    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. 
         [0110]      FIG. 16  reveals a disposable cytometer cartridge  14  containing the pumps, pressure chambers, reservoirs, flow sensors, and a flow mechanism with a flow channel. The flow mechanism may perform hydrodynamic focusing. There might be no external fluid connections on the cartridge. There may be external electrical connections from the cartridge  14  to a controller, computer or processor  40  (hereafter referred to as a processor). However, processor  40  or a portion of it may be included in the cartridge  14 . Processor  40  or a portion of it may be in a form of a chip. External to cartridge  14  may be a light source or sources and detector or detectors associated with the flow channel on the cartridge  14 . All of the liquids are self-contained in the cartridge except for a blood sample that is to be analyzed which is input directly to the cartridge via a port  32 . 
         [0111]    A pump  81  may pump air into a pressure chamber  70 . Pump  81  may be a mesopump as described as an illustrative example by U.S. Pat. No. 5,836,750. Pump  81  may be controlled by processor  40  via a line  89  and connection block  87 . The air may enter a controlled pressure chamber  72  via a valve  74 . The air in chamber  72  may be controlled to be at some pre-determined pressure with mesovalves or other microvalves  74  and  76 . The air may proceed into blood reservoir  62 . Valve  74  may open and valve  76  may close when more air pressure is needed in chamber  72 . Valve  74  may close and valve  76  may open if there is a need to reduce the air pressure in chamber  72 . Valves  74  and  76  may be controlled by processor  40  via line  91  and connection block  60 . Block  60  represents appropriate connections from line  91  to the valves of chamber  72 . The air may proceed through a porous filter  61  on to a blood reservoir  62 . Filter  61  may permit a passage of air but blocks the passage of liquid. The air may exert a controlled pressure on the liquid blood in the reservoir  62 . The blood may flow from the reservoir  62  through flow sensor  80 . Flow sensor  80  may provide information relating to the amount of blood flowing through the sensor via a connection block  48  and line  93  to processor  40 . With the sensed blood flow information, processor  40  may send control signals to valves  74  and  76  to control the air pressure upon the liquid in the reservoir  62  so as to result in a predetermined flow rate of the blood into a flow mechanism  88  which may have a flow channel and hydrodynamic focusing. 
         [0112]    In a similar fashion as for the blood provision, the lyse provision may have a pump  83  that pumps air into a pressure chamber  71 . Pump  83  may be like pump  81 . Pump  83  may be controlled by processor  40  via the line  89  and connection block  87 . The air may enter a controlled pressure chamber  75 . The air in chamber  75  may be controlled to be at some predetermined pressure with valves  84  and  86 . The air may proceed on through a porous filter  63  to a lyse reservoir  64 . Valve  84  may open and valve  86  may close when more air pressure is needed in chamber  75 . Valve  84  may close and valve  86  may open if there is a need to reduce air pressure in chamber  75 . Valves  84  and  86  may be controlled by processor  40  via line  91  and connection block  60 . Block  60  represents an appropriate connection from line  91  to the valves of chamber  75 . The air may proceed through a porous filter  63  on to a lyse reservoir  64 . Filter  63  may permit a passage of air but block the passage of liquid. The air may exert a controlled pressure on the liquid lyse in the reservoir  64 . The lyse may flow from the reservoir through flow sensor  100 . Flow sensor  100  may provide information about the amount of lyse flowing through the sensor via a connecting block  48  and line  93  to processor  40 . With the sensed lyse flow information, processor  40  may send control signals to valves  84  and  86  to control the air pressure upon the liquid in the reservoir  64  so as to result in a predetermined flow rate of the lyse into a flow focusing mechanism  88 . 
         [0113]    In a similar fashion, as for the blood and lyse provisions, the sheath provision may have a pump  85  that pumps air into a pressure chamber  73 . Pump  85  may be a pump like pumps  81  and  83 . Pump  85  may be controlled by processor  40  via the line  89  and connection block  87 . The air may enter from chamber  73  to a controlled pressure chamber  77 . The air in chamber  77  may be controlled to be at some predetermined pressure with valves  94  and  96 . The air may proceed from chamber  77  through a porous filter  65  on to a sheath reservoir  66 . Valve  94  may open and valve  96  may close when more air pressure is needed in chamber  77 . Valve  94  may close and valve  96  may open if there is a need to reduce air pressure in chamber  77 . Valves  94  and  96  may be controlled by processor  40  via line  91  and connection block  60 . Block  60  represents an appropriate connection from line  91  to the valves of chamber  77 . The air may exert a controlled pressure on the liquid sheath in the reservoir  66 . The sheath may flow from the reservoir through flow sensor  102 . Flow sensor  102  may provide information about the amount of sheath flowing through the sensor  102  via a connecting block  48  and line  93  to processor  40 . With the sensed sheath flow information, processor may send signals to pump  85  and valves  94  and  96  to control the air pressure upon the sheath liquid in the reservoir  66  so as to result in a predetermined flow rate of the sheath into the flow mechanism  88 . Ports, connected to an external pressurized air supply, may be implemented in lieu of pumps  81 ,  83  and  85  on cartridge  14  in  FIG. 16 . 
         [0114]    In the flow mechanism  88 , the blood from reservoir  62  may be lysed of its red blood cells and inserted with a flow channel ( 50 ) with a sheath liquid around the stream of the white cells (remaining) in the blood into a single file. The white cells and other particles may be illuminated by light sources, and light from the flow channel may be detected by detectors. The light sources and detectors may be controlled and information may be had from them via connections on line  97  between processor  40  and mechanism  88 . Mechanism  88  and the flow channel are described in other places of the present description. After the blood sample along with the sheath leave the flow channel of mechanism, it may go into the waste reservoir  52 . 
         [0115]    Before the cartridge  14  is used and until its system is pressurized, a set of downstream valves  110  between reservoirs  62 ,  64  and  66  and mechanism  88  may be closed. Their closure and open status may be controlled by processor  40  via line  95  and connection block  110 . 
         [0116]      FIG. 17   a  is another version of the cartridge  14  where all of the liquid devices and operations occur on cartridge.  FIG. 17   b  reveals the same version as that of  FIG. 17   a  except a portion or all of the processor  40  may be situated in the cartridge  14 . Processor  40  in  FIG. 17   b  may communicate externally from the cartridge  14  via line  155 . The cartridge  14  in  FIGS. 17   a  and  17   b  may have a set of valves that are closed to seal off the fluids in the reservoirs  64  and  66  while cartridge  14  is on the shelf. The valves may be mesovalves. Processor  40  may control valves via a line  113  and a connection block  111  providing a connection from the valves to line  113 . Also, the input  32  may be closed off from blood reservoir  62  with a valve  109 . Valve  109  may be a mesovalve or other microvalve connected to processor  40 . While cartridge  14  is on the shelf, downstream valves between the flow sensors and mechanism  88  may be closed. Also, before cartridge  14  is used and until its system is pressurized, the downstream valves may be closed. The closure and open status of the downstream valves may be controlled by processor  40  via line  95  and connection block  110 . Porous filters  61 ,  63  and  65  to reservoirs  62 ,  64  and  65 , respectively, may prevent the passage of liquid but permit the passage of air, assuming the valves of block  111  are open, to enter the reservoirs  62 ,  64  and  66 , so that when the liquids are pumped out the respective reservoirs, the removed liquids may be replaced by air so that a vacuum in not developed in the reservoirs. 
         [0117]    A blood sample may be entered into the blood reservoir  62  via port  32 . A pump  81  may pump blood from reservoir  62  through a flow sensor  80  into the flow mechanism  88 . Flow sensor  80  provides a signal indicating a rate of flow of the blood via connection block  48  and line  93  to processor  40 . Processor  40  may control the amount of flow through sensor  80  by a control signal to pump  81  via line  89  and connection block  87 . 
         [0118]    Lysing liquid in the lyse reservoir  64  may be pumped through flow sensor  100  by a pump  83 . Flow sensor  100  may sent a signal to processor  40 , indicating a rate of flow of the lyse through the sensor into the flow mechanism  88 . This signal may go to processor  40  via the connection block  48  and line  93 . Processor  40  may adjust the rate of flow of the lyse through sensor  100  with a signal sent to pump  83  via line  89  and connection block  87 . 
         [0119]    Sheath fluid may be pumped by pump  85  from the sheath reservoir  66  through a flow sensor  102  on into the flow mechanism  88 . Flow sensor  102  may send a signal indicating the amount of flow of the sheath liquid passing through the sensor  102 . This signal may go to processor  40  via the connection block  48  and line  93 . Processor  40  may adjust the amount or rate of flow of the sheath liquid through sensor  102  and into mechanism  88  with a signal sent to pump  85  via the line  89  and connection block  87 . 
         [0120]    The sample blood may enter mechanism  88  and be lysed with the fluid pumped from reservoir  64  through sensor  100  to remove the red blood cells. The lysed blood may go through a flow channel ( 50 ) with a sheath liquid around the blood causing the white cells in the blood to go through the flow channel in single file. The white cells and other particles may be illuminated with light from the light sources. Light from the flow channel may be detected by detectors. The light sources may be controlled by processor  40  via line  97  to mechanism  88 . The detectors may provide information signals to processor  40  via line  97 . Flow mechanism  88  and the flow channel with its optics are described in other places of the present description. After the blood sample along with the sheath liquid leave the flow channel of the mechanism, it may go into the waste reservoir  52 . 
         [0121]      FIGS. 18   a - 18   d  show a microfluidic cartridge or chip  14  which may be produced with a rapid prototyping, laser-cutting lamination technology. A single type of reagent may be used, but for the convenience of driving, three reagent reservoirs may be included on the fluidic cartridge or chip  14 , together with a waste container  52 . Also, on the chip may be a sample-collecting capillary  32 . The reagent reservoirs  62 ,  64  and  66  may have a pneumatic/hydraulic interface with the cover of the cytometer, which may ensure fluid driving inside of the fluidic chip  13 . The interface may be either a flexible diaphragm or a porous plug (the latter is shown)  61 ,  63  and  65  that may permit air to move through, but prevent fluid loss. Plugs  61 ,  63  and  65  may be located at ports  123 ,  125  and  126 , respectively. As part of a fluid-driving system, flow-sensor dies  80 ,  100  and  102  may be included on the fluidic chip. The electrical connection may be achieved through metal lines deposited on the cartridge  14  and connected to the external holder. 
         [0122]    During storage, a removable cap  114  may be attached to the microfluidic circuit of chip  14 . Lyse may be stored on board in reservoir  64 . Valves  115  and  116  may be open. Valves  117 ,  118 ,  119  and  121  may be closed. One may do an analysis or test on cartridge  14 . 
         [0123]    Cap  114  may be removed and a drop of blood may put into the sample inlet  32 . Capillary action may draw the blood into the sample storage sub-circuit. The cap  114  may be snapped on to the sample inlet  32  and the cartridge  14  may be placed into the cytometer case or bench apparatus. 
         [0124]    By closing the cover of the chip, card or cartridge  14  holder, valves  115  and  116  may close and valves  117 ,  118 ,  119  and  121  may open as in  FIG. 18   b . Reservoirs  62 ,  64  and  66  may be driven by different pressures at ports  123 ,  125  and  126 , respectively, to produce different flow rates in the corresponding fluid lines. Whole blood may be pushed into the sample injector  129  by blood driver/reservoir  62  via valve  121 , line  128 , valve  117  and line  127  at a flow rate of approximately 0.1 microliter per second. In parallel, lyse from reservoir  64  may be pushed, via valve  118  and line  131  of  FIG. 18   b , on to sample injector  129  at a flow rate of approximately 1 microliter per second. In  FIG. 18   c , the lyse and whole blood may be co-eluded into a mixing and lysing channel  133  to produce a total of about 13 microliters of about 10 to 1 diluted blood (viz., the sample). The red blood cells are lysed, leaving white blood cells remaining in the sample. In  FIG. 18   d , a sheath fluid may be pushed via valve  119  and line  132  into a focusing chamber  134  at a rate of about 7 microliters per second. Blood flow may be stopped with the reduction of the pressure load in reservoir  62  to zero, while the pressure load in reservoir  64  is adjusted to produce a sample (lysed blood) at a flow rate of about 0.5 microliter per second. The sheath fluid in chamber  134  may cause the while cells of the blood sample to be hydrodynamically focused or the like in area  135  into single file core stream to flow through flow channel  50 . These flow rates may be needed for producing a core stream with dimensions of about 10×5 microns in the cytometer flow channel  50 . 
         [0125]    The particles or cells in flow channel  50  may pass by the light source and detector system  136 . Small angle scattering (SALS), forward angle scattering (FALS) and large angle scattering (LALS) caused by the particles in the flow stream may be detected. Arrays of light sources and detectors may be used. Also, interruptions of direct light may be detected. Particle width, length, center and velocity may be determined. Various other properties and identification information of the particles may be obtained with the optical system. 
         [0126]    A previous cytometer system used so-called volume-controlled flow, generated by miniature syringe pumps driven with stepper motors or manually, to drive all reagents and the sample through the microfluidic circuit of cartridge  14 . The system may be precise but is extremely bulky and uses significant electrical power. In order to miniaturize and make energy efficient the fluid driving system, the open-loop, very precise and stable but bulky and expensive fluid driving elements may be replaced with less precise and less stable pressure sources which can be adjusted in a closed loop configuration to maintain a constant, desired flow velocity at critical points of the fluidic circuit. Implementation of this approach may rely on small and sensitive fluid flow sensors for measurement of flow rates as low as 10 nanoliters per second in sub-millimeter channels, and fast and small actuators for closed loop, pressure control. 
         [0127]    There may be a manually pressurized system described in other places of this description. Another approach may involve active pumping accomplished with mesopump channels.  FIGS. 19   a  and  19   b  reveal an application of the mesopumps  137  and mesovalves  138  embedded in a chip, card or cartridge  14 . There may also be embedded flow sensors  139  in chip  14 .  FIGS. 19   a  and  19   b  show an illustrative example of a portion of a fabricated chip or cartridge  14  with the embedded components.  FIG. 19   b  is a top view of the portion of a cytometer and  FIG. 19   a  is a cut away side view revealing the structural relationship of the components relative to the chip  14 . Configurations of the cartridges as shown in  FIGS. 16 ,  17   a  and  17   b , as illustrative examples, may have embedded mesopumps  137  as pumps  81 ,  83  and  85 , and mesovalves  138  as the valves in blocks  110  and  111  of  FIGS. 16 ,  17   a  and  17   b , and mesovalves  138  as valves  74 ,  76 ,  84 ,  86 ,  94  and  96  of pressure chambers  72 ,  75  and  77  of  FIG. 16 . Other valves in the system may be embedded mesovalves  138 . Similarly, valves  115 - 119  and  121  of the cartridge  14  of  FIGS. 18   a - 18   d  may be embedded mesovalves  138 . Flow sensors  80 , 100  and  102  of cartridge  14  of  FIGS. 16 ,  17   a ,  17   b ,  18   a - 18   d ,  19   a  and  19   b  may be embedded flow sensors  139 . However, the pumps and valves may be another kind of small valves and pumps. 
         [0128]    Mesopumps  137  may be, for an example, dual diaphragm pumps which are in principle described in U.S. Pat. No. 6,179,586 B1, issued Jan. 30, 2001, which is incorporated herein by reference. Also, information related to mesopumps and valves may be disclosed in U.S. Pat. No. 5,836,750, issued Nov. 17, 1998, which is incorporated herein by reference. U.S. Pat. Nos. 6,179,586 B1 and 5,836,750 are owned by the entity that owns the present invention. 
         [0129]      FIGS. 20   a  and  20   b  reveal an illustrative example of a mesovalve  141  in a closed state and an open state, respectively. In  FIG. 20   a , there may be a diaphragm  142  closing off an output port  149  at the valve-like seat  144  of a lower structure  145 . Diaphragm  142  may have a first electrode  146  coated on it. Surfaces of an inside cavity  151  of a top structure  148  may have a second electrode  147  coated on them. Lower structure  145  may have an input port  143  to the mesovalve  141 . Diaphragm  142  may seal the output port  149  from the tension of diaphragm  142  being held between the upper and lower structures. The valve seat  144  upper surface may be slightly higher than the surface of the perimeter of the lower structure  145  securing the diaphragm  142 . Also, there may be a repelling electrostatic force between electrodes  146  and  147  pushing diaphragm  102  against the valve-like seat  144 . 
         [0130]    In  FIG. 20   b , diaphragm  142  may be lifted off of valve-like seat  144  with an attracting electrostatic force between the electrode  146  attached to diaphragm  142  and electrode  147  adhered to the inside surfaces of top structure  148 . With diaphragm  142  lifted off of surface or seat  144 , a fluid  153  may flow from the import port  143  in a cavity  153  below diaphragm  142 , through the cavity, and past the seat surface  144  into the output port  149 . The electrostatic force attracting electrodes  146  and  147  may be caused by an application of an electrical voltage to the electrodes  146  and  147 . When the electrical voltage across the electrodes  146  and  147  is removed, electrostatic attraction between diaphragm  142  and the inside surfaces of top structure  143  disappear, diaphragm  142  may fall and return its original position against surface  144  which seals off the output port  149  to stop the flow of fluid  153  through the mesovalve  141 . 
         [0131]      FIG. 21  shows a fluid micro or mesovalve  159  situated or embedded in the cartridge  14 , having an off-cartridge controller  40  connected to the valve. Valve  159  may be another kind of valve situated in the cartridge.  FIG. 22  shows the cartridge  14  having a fluid pump  158  embedded or built into it. Pump  158  may provide unidirectional or bidirectional flow. The pump may be a mesopump or other kind of a pump. It may be utilized for gas or liquid. Pump  158  may be open-loop controlled by control  40 . Control  40  may be a processor and/or a controller. The present pumps and valves discussed in this description may have thicknesses from 0.8 mm to 1.0 mm, which might be reduced. However, the pumps and valves may be as thin as 0.2 mm. Application of various technologies may reduce the thicknesses even further. The pumps and valves may have diameters of about 10 mm. The pumps and valves may be stacked upon each other whether they are connected to one another or not. The range of thicknesses of cartridge  14  may be about 1 mm to 5 mm, i.e., generally a thickness less than 6 mm, but should be less than 10 mm, although it could be thinner than 1 mm. Lateral dimensions of the cartridge  14  may be less than 5 cm by 7 cm, but should be less than 10 cm by 15 cm, or an area less than 150 square cm. The cartridge could be about the size of a typical credit card. In certain applications, the cartridge may be thicker than 10 mm and/or larger than 10 cm by 15 cm or an area larger than 150 square cm. This larger size cartridge may encompass much more complex microfluidics. The pumps and valves may be encompassed in the cartridge  14  using laminate technologies. There may be embedded components such as pumps and valves that are put into the cartridge or built in as a part of the layers of the cartridge. The cartridge  14  may also have other components including flow sensors, pressure sensors, passage ways, devices for preventing a flow of liquid, channels and reservoirs for fluids and their flow. These components may be micro-components, including mesopumps and mesovalves. The pumps may be unidirectional or bidirectional. Some of these components may be situated on the cartridge and some may be situated off the cartridge  14 . The combination of on-cartridge and off-cartridge components may vary according to application of the cartridge. Not all combinations of on-cartridge and off-cartridge components are necessarily shown in the Figures of the present description. The cartridge  14  may be treated as a disposable or non-disposable item after usage. When used for blood analysis, the cartridge  14  would likely be disposed of for sanitary reasons. If the cartridge is used for water, environmental, pollutant or like analysis, the cartridge  14  may be reusable. 
         [0132]    The flow of fluid in the configuration of  FIG. 22  may be determined by noting the number of cycles of pump  158  per volume unit of flow. The flow amount may be set by control  40  of the pump.  FIG. 23  shows a liquid pump  161  on the cartridge  14 . Pump  161  may be unidirectional or bidirectional. A liquid may be pumped by pump  161  in either direction past a liquid flow sensor  163 . The sensor  163  may also be embedded or built into cartridge  14 . Flow sensor  163  may provide a feedback signal to control  40  to indicate the amount and direction of flow. Control  40  may maintain a closed loop control of pump  161  so as to provide a specific flow on cartridge  14 .  FIG. 24  shows a similar type of fluid circuit as that in  FIG. 23 , except it shows a gas pump which may be designed to pump a gas in one or two directions in cartridge  14 . The pumps in the various configurations of this description may be a mesopumps or other kinds of micropumps. Also, the valves of the configurations may be mesovalves or other kinds of microvalves. Some of these pumps may pump both gas and liquid. A gas flow sensor  164  may indicate a direction and an amount of flow on cartridge  14 . A gas flow indication may be sent to control  40  which in turn provides an input to pump  162  so as to provide a desired flow. 
         [0133]      FIG. 25  shows another pumping configuration for providing or removing a liquid from a reservoir  165  on a cartridge  14 . The pump  162 , open loop controlled by control  40 , may provide gas through a buffer  166  (which may be like a pressure chamber) to apply pressure to the reservoir  165 . Buffer  166  may smooth out the pulsations in the gas flow caused by gas pump  162 . There may be a device or membrane  157  that may permit gas but not liquid to go through it. The buffer  166  may not be needed in some configurations. The gas may go to a liquid reservoir  165  and with a build up of pressure of the gas on the liquid in the reservoir  165  to push out the liquid from the reservoir.  FIG. 26  shows a similar configuration not on a cartridge  14 . Certain portions of this configuration may be on or off of the cartridge  14 . 
         [0134]      FIG. 27  is similar to  FIG. 25  except that the configuration of the  FIG. 27  has a closed loop control with a flow sensor  163 . The gas pump  162  may pump a gas to liquid reservoir  165  via buffer  166 . Pump  162  may be unidirectional or bidirectional. Gas to the reservoir  165  may provide pressure on the fluid to move it from the reservoir  165  through the liquid flow sensor  163 . A device  157  may prevent liquid from flowing back into buffer  166  but will let gas go through in either direction. The flow sensor  163  may send a signal to control  40  indicating an amount of liquid flow from the reservoir. Control  40  may provide a signal to gas pump  162  to control the amount of gas pumped so as to maintain an appropriate gas pressure and/or desired flow of liquid from the reservoir  163 , via the closed loop connections to and from control  40 .  FIG. 28  reveals a similar configuration of the one in  FIG. 27 , except that it may be wholly or partially off of the cartridge  14 . 
         [0135]      FIG. 29  reveals another configuration that may be inserted in cartridge  14 . A gas  162  pump may pump a gas through a buffer  166  and onto a pressure chamber  167  at an input having a valve  168 . Also, chamber  167  may have a relief-like valve  169 . Valves  168  and  169  may be actuated by control  40  to open or close individually. The pump  162  may be unidirectional or bidirectional. The gas may proceed from chamber  167  to the liquid reservoir  165  where a pressure of the gas on the liquid in reservoir may force the liquid through the liquid flow sensor  163 . Flow sensor  163  may be off of the cartridge  14  but still coupled to the microfluidic circuit on the cartridge. Flow sensor  163  may send a signal indicating flow to control  40 . Control  40  may assure that pump  162  is pumping sufficient gas. However, the amount of liquid flow through flow sensor  163  may be controlled by an amount of pressure in chamber  167 . If more pressure is needed for increased flow through sensor  163 , control  40  may open valve  168  and close valve  169 . If the pressure needs to be decreased to reduce liquid flow through sensor  163 , then control  40  may close valve  168  and open valve  169 . Valves  168  and  169  may be mesovalves embedded or built into the cartridge  14 . The closed loop control may be limited to just the flow sensor and valve operation. Pump  162 , in either configuration, may be a mesopump or other micropump.  FIG. 30  shows another configuration on cartridge  14  having some resemblance to the configuration of  FIG. 29 ; however, the flow sensor  163  may be on the cartridge  14 . 
         [0136]      FIG. 31  shows a configuration having a pressure chamber  176  being controlled differently than chamber  167  of  FIGS. 29 and 30 . A gas pump  162  may pump gas through a buffer  166  on to a pressure chamber  171  via a valve  172 . The gas under pressure may go to liquid reservoir  165 . The gas on the liquid in the reservoir may force the liquid out of the reservoir on through the flow sensor  163 . A signal indicating liquid flow may be sent from sensor  163  to control  40 . Control  40  may assure that pump  162  is pumping a sufficient amount of gas. However, the amount of liquid flow through flow sensor may be controlled by an amount of pressure on the gas in chamber  167 . The amount of pressure may be detected by pressure sensor  173  in chamber  171 , and a signal indicating the amount of pressure may be sent from sensor  173  to control  40 . If more flow of liquid is to go through flow sensor  163 , then valve  172  may be at least partially opened; and if less flow of liquid is to go through the flow sensor, then valve  172  may be moved into a more closed position but not necessarily be completely closed. The opening and closure of valve  172  may be controlled by signals from control  40 . Instead of a relief valve  169  as in  FIG. 31 , a restrictor  174  may be placed in the pressure chamber  171  to provide some leakage or relief of gas from chamber  171 . Valve  172  may be a mesovalve embedded or built into the cartridge  14 . Similarly, the restrictor or orifice  174  may be built into cartridge  14 . Pressure sensor  173  may be embedded of built into cartridge  14 .  FIG. 32  may have a similar configuration as that of  FIG. 31 , except that the configuration of  FIG. 32  is shown as off of the cartridge. Also,  FIG. 33  may have a similar configuration as that of  FIG. 30 , except that the latter is shown as off of the cartridge. Certain portions of either configuration may be on or off the cartridge. 
         [0137]    The configurations of  FIGS. 29-33  may have several closed loop control arrangements which may be implemented separately or in combination. The flow sensor and gas pump in conjunction with each other may provide sufficient closed loop control. The pressure chamber and its valves with a flow sensor may provide sufficient closed loop control. In  FIGS. 31 and 32 , the pressure chamber with the pressure sensor and valve may separately provide sufficient closed loop control. 
         [0138]    Although the some of the components discussed in  FIGS. 21-33 , except control  40 , may reside on the cartridge  14 , some of the components may be located off of the cartridge. Further, control  40  may be a chip embedded or built into the cartridge  14 , although control  40  or a portion of it may often be located off of the cartridge. In FIGS.  27  and  29 - 31 , gas pump  162  and/or buffer  166  may be located off the cartridge  14 . They may be connected via ports and tubing or other plumbing to cartridge  14  when the cartridge is placed into a holder that facilitates the fluid and electrical connections and optical interface for cartridge  14 . The cartridges  14 , as in  FIGS. 21-25 ,  27  and  29 - 31 , may be shown merely in part. These cartridges may have additional components relevant to specific applications. 
         [0139]    Liquid flow sensor  163  may be embedded or built into the cartridge  14  or it may be removed from the cartridge which may be disposed of after usage, and reused in another cartridge  14 . Liquid flow sensor  163  may be may be removable from a slot or holder in one cartridge  14  for reusability in another cartridge with a similar slot or holder. The same may apply to the gas flow sensor  164 . The various off-cartridge components may be connected to the components on the cartridge  14  via appropriate plumbing. 
         [0140]    Even though the  FIGS. 21-33  show one channel for cartridge  14 , a cartridge  14  may have two or more channels having similar or differing configurations. Also, cartridge  14  may have a flow channel or like mechanism associated with the configurations of  FIGS. 21-33 . A flow channel and additional components may be on the cartridge  14  even for configurations in the Figures revealing some or all of the components shown in the respective Figures as being off the cartridge. 
         [0141]    In the present specification, some of the matter may be of a hypothetical or prophetic nature although stated in another manner or tense. 
         [0142]    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.

Technology Classification (CPC): 1