Abstract:
Determining a biological condition in a mammal by using a cartridge, having fluidic open channels, sealable after receiving a fluid specimen, passing any of the specimen through any of the channels, contacting any reagent stored in a chamber with the specimen in a reaction chamber inducing a reaction and forming a reaction product, a mechanical controller including first urging means applying a force externally onto the chamber to release the reagent, second urging means applying a removable force onto the channels thereby inducing fluidic movement in a first direction in the channels and upon removal of the force causing fluidic movement in an opposite direction, alignment means aligning a reading channel on the cartridge for a detection to take place, an optical reader detecting the reaction product in the reading channel, and a processor receiving data from the optical reader and processing the data to determine the biological condition.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation-in-part of U.S. patent application Ser. No. 13/716,246, filed on Dec. 17, 2012, and claims priority from U.S. Provisional Patent Application Nos. 61/737,854, to Kasdan, et al., filed on Dec. 17, 2012, and 61/737,856, to Kasdan, et al., filed on Dec. 17, 2012, the disclosures of which are incorporated herein by reference in their entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates generally to apparatus and methods for detecting a biological condition, and more specifically to methods and apparatus for detecting a biological condition in small fluid samples. 
       BACKGROUND OF THE INVENTION 
       [0003]    There are numerous medical conditions which are hard to diagnose. Often diagnosis by a physician is based on the physician&#39;s observation of combinations of symptoms in a patient. This sometimes leads to misdiagnosis. Furthermore, the patient&#39;s response to a treatment, whether drug or other modality is often followed up by physician&#39;s observation. 
         [0004]    Many laboratory tests are performed in the diagnostic arena on a bodily specimen or fluid to determine a biological condition in a patient. However, these tests are performed off-line in diagnostic laboratories. Often, the laboratory services are only provided during a single 8-hour shift during the day and tend to be labor intensive. Some prior art publications in the field include, inter alia, U.S. Pat. No. 8,116,984, US2006215155 and US2012187117. 
         [0005]    Despite the inventions mentioned hereinabove, there still remains an unmet need to provide improved apparatus and methods for detecting and diagnosing biological conditions in a patient. 
       SUMMARY OF THE INVENTION 
       [0006]    It is an object of some aspects of the present invention to provide improved apparatus and methods for detecting and diagnosing biological conditions in a patient. 
         [0007]    In some embodiments of the present invention, improved methods, apparatus and systems are provided for detecting and diagnosing a biological condition in a patient. 
         [0008]    In other embodiments of the present invention, a method and system is described for providing rapid detection of biological moieties in a sample from a patient. 
         [0009]    In further embodiments of the present invention, a method and system is disclosed for providing detection of biological moieties in a small fluid sample from a patient. 
         [0010]    There is thus provided according to an embodiment of the present invention, a system for determining a biological condition, the system including;
       a) a cartridge including a plurality of fluidic open channels, all the channels in liquid communication with each other, the cartridge being adapted to be sealed after receiving a fluid specimen and to pass a predetermined quantity of the specimen through at least part of the plurality of fluidic open channels; and further to vigorously contact at least one reagent stored in a sealed on-board chamber (termed herein “a blister”) with the predetermined quantity of the specimen in a reaction chamber to induce a reaction and form a reaction product;   b) a mechanical controller including;
           i. a first urging means adapted to apply a force externally onto the chamber thereby breaking a frangible seal on the chamber and to release the at least one reagent;   ii. at least one second urging means adapted to apply a removable force onto plurality of fluidic open channels thereby inducing fluidic movement in a first direction in the plurality of fluidic open channels and upon removal of the force causing fluidic movement in an opposite direction to the first direction;   iii. an alignment means adapted to align a reading channel on the cartridge for a detection to take place;   
           c) an optical reader adapted to detect the reaction product in the reading channel of the sealed cartridge; and   d) a processor adapted to receive data from the optical reader and to process the data to determine the biological condition;       
 
         [0018]    wherein the cartridge further includes an inflatable deformable elastic chamber (termed herein “a bellows”) adapted to apply at least one of a negative pressure and a positive pressure in the fluidic channels. 
         [0019]    Additionally, according to an embodiment of the present invention, the plurality of fluidic open channels is of a cross-section of 0.1 to 2 mm 2 . 
         [0020]    Furthermore, according to an embodiment of the present invention, the fluid specimen is of a volume of 10 to 500 microliters. 
         [0021]    Importantly, according to an embodiment of the present invention, the cartridge is valveless. 
         [0022]    Moreover, according to an embodiment of the present invention, the cartridge limits processing of the fluid specimen to a fixed volume that is a fraction of the input volume. 
         [0023]    Further, according to an embodiment of the present invention, the cartridge is a closed system after sealing. 
         [0024]    Yet further, according to an embodiment of the present invention, the cartridge is adapted to contact a plurality of on-board reagents with at least one of the specimen and the reaction product. 
         [0025]    Additionally, according to an embodiment of the present invention, the cartridge is adapted to induce cascaded sequential reactions of the on-board reagents with at least one of the specimen and the reaction product. 
         [0026]    Furthermore, according to an embodiment of the present invention, the reaction chamber is of a volume of 200 to 10000 microliters. 
         [0027]    Moreover, according to an embodiment of the present invention, the system further includes a temperature control device external to the cartridge, the device being adapted to control wherein the cartridge is adapted to contact a temperature of the reaction. 
         [0028]    Importantly, according to an embodiment of the present invention, the cartridge has a shelf-life of 6 to 24 months. 
         [0029]    Notably, according to an embodiment of the present invention, the cartridge is static within the system. 
         [0030]    Additionally, according to an embodiment of the present invention, the fluid specimen is introduced to the cartridge via capillary action. 
         [0031]    Furthermore, according to an embodiment of the present invention, the cartridge includes at least one of the following elements;
       i. a reservoir;   ii. a pump;   iii. a conduit;   iv. a miniaturized flow cell;   v. a transport channel;   vi. a reading channel;   vii. a microfluidic element;   viii. a compressed gas holding element   ix. a compressed gas releasing element;   x. a nozzle element;   xi. a mixing element;   xii. a bellows element.   xiii. software adapted to activate the elements according to a specific sequence; and   xiv. hardware to activate the elements according to a specific sequence.       
 
         [0046]    Further, according to an embodiment of the present invention, the cartridge includes several of the above-mentioned elements. 
         [0047]    Yet further, according to an embodiment of the present invention, the cartridge includes all of the above-mentioned elements. 
         [0048]    Additionally, according to an embodiment of the present invention, the at least one composition disposed in the cartridge includes at least one of;
       a. at least one target antibody;   b. at least one positive control identifying antibody; and   c. at least one negative control identifying detection moiety.       
 
         [0052]    Additionally, according to an embodiment of the present invention, the at least one composition disposed in the cartridge includes two of:
       a. at least one target antibody;   b. at least one positive control identifying detection moiety; and   c. at least one negative control identifying detection moiety.
 
In some cases, according to an embodiment of the present invention, the at least one composition disposed in the cartridge includes all of:
   a. at least one target antibody;   b. at least one positive control identifying detection moiety; and   c. at least one negative control identifying detection moiety.       
 
         [0059]    Additionally, according to an embodiment of the present invention, the at least one composition disposed in the cartridge includes at least one reference composition including at least one of;
       d. a target signal reference composition; and   e. a reference identifier composition.       
 
         [0062]    Furthermore, according to an embodiment of the present invention, the at least one composition disposed in the cartridge includes at least one of;
       f. a positive control moiety; and   g. a negative control moiety.       
 
         [0065]    Moreover, according to an embodiment of the present invention, the at least one composition disposed in the cartridge includes a sepsis biomarker. 
         [0066]    Additionally, according to an embodiment of the present invention, the biomarker includes at least one of CD64 and CD163. 
         [0067]    There is thus provided according to an additional embodiment of the present invention, a method for determining a biological condition in a subject, the method including;
       a. incubating a specimen from the subject in the system of as described herein for a predetermined period of time; and   b. receiving an indication responsive to the at least one reporter element thereby providing the indication of the biological condition in the subject.       
 
         [0070]    Additionally, according to an embodiment of the present invention, the biological condition is selected from blood diseases such as leukemia, thrombocytopenia, immune system disorders, local infections, urinary tract disorders, autoimmune diseases and sepsis. 
         [0071]    Importantly, according to an embodiment of the present invention, the indication is quantitative. 
         [0072]    Additionally, according to an embodiment of the present invention, the method is completed within twenty minutes. 
         [0073]    Notably, according to an embodiment of the present invention, the system is a flow cytometer system. 
         [0074]    There is thus provided according to an additional embodiment of the present invention, a method for determining a biological condition in a mammalian subject, the method including;
       a. incubating a specimen from the subject with at least one composition in a system for a predetermined period of time to form at least one reaction product, when the subject has the biological condition; and   b. receiving an indication of the at least one reaction product responsive to at least one reporter element in the system thereby providing the indication of the biological condition in the subject.       
 
         [0077]    There is thus provided according to an additional embodiment of the present invention, an automated method of determining the presence or absence of sepsis in a subject, including;
       a. contacting a blood sample from the subject with a fluorescently-labeled binding moiety specific to a sepsis marker, wherein the volume of the blood sample is 50 μL or smaller;   b. detecting the presence, absence or level of the binding moiety in the sample, thereby determining the presence or absence of sepsis in the subject within twenty minutes.       
 
         [0080]    Additionally, according to an embodiment of the present invention, the sepsis marker is CD64. 
         [0081]    Furthermore, according to an embodiment of the present invention, the sepsis marker is CD163. 
         [0082]    Moreover, according to an embodiment of the present invention, the method further includes contacting the blood sample with a second fluorescently-labeled binding moiety specific for a second sepsis marker. 
         [0083]    Additionally, according to an embodiment of the present invention, the sepsis marker is CD64 and the second sepsis marker is CD163. 
         [0084]    There is thus provided according to an embodiment of the present invention, a system for evaluating a biological condition in a patient, the system comprising;
       a) a disposable element for receiving a biological specimen and for combining said specimen with at least one composition;   b) at least one composition comprising at least one detector moiety adapted to react with said specimen to form a reaction product, when said patient has said biological condition; and   c) at least one reporter element adapted to provide an indication of reaction product thereby providing the indication of the biological condition.       
 
         [0088]    Additionally, according to an embodiment of the present invention, the system further comprises;
       d) instructions for using the system.       
 
         [0090]    Furthermore, according to an embodiment of the present invention, the disposable element is a disposable cartridge. 
         [0091]    Moreover, according to an embodiment of the present invention, the disposable cartridge is a disposable microfluidics cartridge. 
         [0092]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least one of the following elements: 
         [0093]    a) a reservoir; 
         [0094]    b) a pump; 
         [0095]    c) a valve; 
         [0096]    d) a conduit; 
         [0097]    e) a motor; 
         [0098]    f) a miniaturized flow cell; 
         [0099]    g) a transport channel; 
         [0100]    h) a microfluidic element; 
         [0101]    i) a compressed gas holding element; 
         [0102]    j) a compressed gas releasing element; 
         [0103]    k) a nozzle element; 
         [0104]    l) a mixing element; 
         [0105]    m) a bellows element; 
         [0106]    n) software adapted to activate said elements according to a specific sequence; and 
         [0107]    o) hardware to activate said elements according to a specific sequence. 
         [0108]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least two of the elements. 
         [0109]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least three of the elements. 
         [0110]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least four of the elements. 
         [0111]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least five of the elements. 
         [0112]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least ten of the elements. 
         [0113]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least twenty of the elements. 
         [0114]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least thirty of the elements. 
         [0115]    According to an embodiment of the present invention, the microfluidics system is configured to provide the rapid indication with one hour. 
         [0116]    According to another embodiment of the present invention, the microfluidics system is configured to provide the rapid indication with thirty minutes. 
         [0117]    According to another embodiment of the present invention, the microfluidics system is configured to provide the rapid indication with fifteen minutes. 
         [0118]    According to another embodiment of the present invention, the microfluidics system is configured to provide the rapid indication with ten minutes. 
         [0119]    According to another embodiment of the present invention, the microfluidics system is configured to provide the rapid indication with five minutes. 
         [0120]    According to another embodiment of the present invention, the microfluidics system is configured to provide the rapid indication with one minute. 
         [0121]    According to another embodiment of the present invention, the microfluidics system is configured to provide the rapid indication with thirty seconds. 
         [0122]    According to another embodiment of the present invention, the microfluidics system is configured to provide the rapid indication with ten seconds. 
         [0123]    According to another embodiment of the present invention, the microfluidics system is configured to provide the rapid indication with one second. 
         [0124]    There is thus provided according to an embodiment of the present invention, a microfluidics assay system for performing a rapid biological assay, the system comprising;
       a) a disposable element comprising a reactant, the disposable element being adapted to receive a sample comprising a biological entity and for combining said reactant with said biological entity to form a reaction product; and   b) at least one reporter element adapted to provide a rapid indication of disappearance of said reactant thereby providing rapid assay of the biological entity.       
 
         [0127]    There is thus provided according to an embodiment of the present invention, a microfluidics assay system for performing a rapid assay of a biological entity, the system comprising;
       a) a disposable element comprising a reactant, the disposable element being adapted to receive a sample comprising the biological entity and for combining said reactant with said biological entity to form a reaction product; and   b) at least one reporter element adapted to provide a rapid indication of appearance of said reaction product thereby providing rapid assay of the biological entity.       
 
         [0130]    There is thus provided according to an embodiment of the present invention, a composition for evaluating a biological condition, the composition comprising;
       a. a sample composition comprising at least one of;
           i. a bodily specimen comprising a target moiety;   ii. a positive control moiety; and   iii. a negative control moiety;   
           b. a detection composition comprising at least one of;
           i. at least one target antibody;   ii. at least one positive control identifying antibody; and   iii. at least one negative control identifying detection moiety or characteristic; and   
           c. at least one reference composition comprising at least one of;
           i. a target signal reference composition; and   ii. a reference identifier composition.   
               
 
         [0142]    There is thus provided according to another embodiment of the present invention a composition for evaluating a biological condition, the composition comprising;
       a. a sample composition comprising at least one of;
           i. a bodily specimen comprising a target moiety;   ii. a positive control moiety; and   iii. a negative control moiety;   
           b. an antibody composition comprising at least one of;
           i. at least one target antibody (CD64 antibody);   ii. at least one positive control identifying antibody (CD  163 ); and   iii. at least one negative control identifying antibody or characteristic; and   
           c. at least one reference composition comprising at least one of;
           i. a target signal reference composition; and   ii. a reference identifier composition.   
               
 
         [0154]    Additionally, according to an embodiment of the present invention, the composition further comprises at least one conditioning moiety comprising;
       d. at least one lysis reagent; and   e. at least one diluent.       
 
         [0157]    Furthermore, according to an embodiment of the present invention, the biological condition is selected from a group consisting of blood diseases such as leukemia, thrombocytopenia immune system disorders, local infections, urinary tract disorders, autoimmune diseases and sepsis. 
         [0158]    Moreover, according to an embodiment of the present invention the bodily specimen is selected from a group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid (CSF), serous fluid, peritoneal fluid and synovial fluid. 
         [0159]    According to another embodiment of the present invention, the target moiety includes a CD64 surface antigen on neutrophils. 
         [0160]    Additionally, according to a further embodiment of the present invention, the positive control moiety includes monocytes and the negative control includes lymphocytes. 
         [0161]    Additionally, according to an embodiment of the present invention, the target moiety is CD64 on neutrophils, the positive control moiety includes CD64 expression on monocytes, and the negative control moiety includes lymphocytes without CD64 expression. 
         [0162]    Further, according to an embodiment of the present invention, the target indicator is bound to a signaling moiety on the at least one target antibody. 
         [0163]    Yet further, according to an embodiment of the present invention, the at least one reference composition includes beads. 
         [0164]    Additionally, according to an embodiment of the present invention, the beads include polystyrene microbeads. 
         [0165]    Moreover, according to an embodiment of the present invention, the target antibody reference composition includes a first fluorescent signal and the reference identifier composition includes a second fluorescent signal. 
         [0166]    Furthermore, according to an embodiment of the present invention, the first fluorescent signal includes FITC and the second fluorescent signal includes Starfire Red fluor. 
         [0167]    There is thus provided according to an embodiment of the present invention, a method of quantifying a biomarker in a sample, comprising;
       a. contacting the sample with a fluorescently-labeled binding moiety that specifically binds to the biomarker;   b. detecting a first fluorescent signal from at least a portion of the labeled sample;   c. detecting a second fluorescent signal from a population of fluorescently-labeled particles, wherein the population includes a known fluorescent intensity over a fixed time; and   d. normalizing the first fluorescent signal to the second fluorescent signal, thereby quantifying the biomarker, wherein the normalizing includes using a device comprising software capable of comparing the first and second fluorescent signal.       
 
         [0172]    Furthermore, according to an embodiment of the present invention, the biomarker is a sepsis biomarker. 
         [0173]    Moreover, according to an embodiment of the present invention, the biomarker is CD64 or CD163. 
         [0174]    Additionally, according to an embodiment of the present invention, the sample is a blood sample. 
         [0175]    According to another embodiment of the present invention, the fluorescent label of the binding moiety and the fluorescent label of the particles is the same fluorescent label. 
         [0176]    Further, according to an embodiment of the present invention, the binding moiety is an antibody. 
         [0177]    According to an embodiment of the present invention, the software is capable of recognizing a specific lot of fluorescently-labeled particles. 
         [0178]    Moreover, according to an embodiment of the present invention, the individual fluorescent signals include at least one first fluorescent signal and at least one second fluorescent signal. 
         [0179]    Additionally, according to an embodiment of the present invention the fluorescently-labeled binding moiety targets a first cell population and a second cell population in the sample. 
         [0180]    According to another embodiment of the present invention the detection of binding of the binding moiety to the second cell population provides an internal positive control for the sample. 
         [0181]    Furthermore, according to an embodiment of the present invention, the binding moiety is anti-CD64 antibody and the first cell population includes neutrophil leukocytes. 
         [0182]    Yet further, according to an embodiment of the present invention, the second cell population includes monocytes. 
         [0183]    According to an embodiment of the present invention, the method further comprises the step of determining the presence of at least one cell population in the sample that is not bound by the binding moiety, thus providing an internal negative control for the sample. 
         [0184]    There is thus provided according to another embodiment of the present invention, a composition for evaluating a biological condition, the composition comprising;
       a. a sample comprising at least one of;
           i. a bodily specimen comprising a target moiety;   ii. a positive control moiety; and   iii. a negative control moiety;   
           b. an antibody composition comprising at least one of;
           i. at least one target antibody;   ii. at least one positive control identifying antibody; and   iii. at least one negative control identifying antibody or characteristic; and   
           c. at least one reference composition comprising at least one of;
           i. a target antibody reference composition; and   ii. a reference identifier composition.   
               
 
         [0196]    According to an embodiment of the present invention, the composition further comprises at least one conditioning moiety comprising;
       a) at least one lysis reagent; and   b) at least one diluent.       
 
         [0199]    There is thus provided according to another embodiment of the present invention, a method of determining the presence or absence of sepsis in a subject, the method including;
       a) contacting a blood sample from the subject with a fluorescently-labeled binding moiety specific to a sepsis marker, wherein the volume of the blood sample is 50 μL or smaller; and   b) detecting the presence, absence or level of the binding moiety in the sample, thereby determining the presence or absence of sepsis in the subject.       
 
         [0202]    There is thus provided according to another embodiment of the present invention, a method of quantifying a biomarker in a sample, comprising;
       a) contacting the sample with a fluorescently-labeled binding moiety that specifically binds to the biomarker;   b) detecting a first fluorescent signal from at least a portion of the labeled sample;   c) detecting a second fluorescent signal from a population of fluorescently-labeled particles, wherein the population includes a known fluorescent intensity over a fixed time; and   d) normalizing the first fluorescent signal to the second fluorescent signal, thereby quantifying the biomarker, wherein the normalizing includes using a device comprising software capable of comparing the first and second fluorescent signal.       
 
         [0207]    According to some embodiments, the sample may be liquid, according to other embodiments, the sample may be a colloid or suspension. According to further embodiments, the sample may be a solid, such as in a powder or crystal form. 
         [0208]    Typical turnaround times for diagnostic prior art assays are 30-120 minutes. Often, the time lost in waiting for laboratory results can lead to a further deterioration in a patient, and sometimes death. In some cases, the physician has to act without having the laboratory results. This can lead to providing the patient with the wrong treatment. The present invention provides rapid assays to save lives and provide fast correct treatments to a patient. 
         [0209]    There is thus provided according to an embodiment of the present invention automated method of determining the presence or absence of sepsis in a subject, including;
       a) contacting a blood sample from the subject with a fluorescently-labeled binding moiety specific to a sepsis marker, wherein the volume of the blood sample is 50 μL or smaller; and   b) detecting the presence, absence or level of the binding moiety in the sample, thereby determining the presence or absence of sepsis in the subject within twenty minutes.       
 
         [0212]    Importantly, according to an embodiment of the present invention, the method is a flow cytometric method. 
         [0213]    Additionally, according to an embodiment of the present invention, the sepsis marker is CD64. 
         [0214]    Furthermore, according to an embodiment of the present invention, a second sepsis marker is CD163. 
         [0215]    Moreover, according to an embodiment of the present invention, the method further includes contacting the blood sample with a second fluorescently-labeled binding moiety specific for a second sepsis marker. 
         [0216]    Further, according to an embodiment of the present invention, the sepsis marker is CD64 and the second sepsis marker is CD163. 
         [0217]    Additionally, according to an embodiment of the present invention, the binding moiety is an antibody. 
         [0218]    Moreover, according to an embodiment of the present invention, the detecting step is performed in a device capable of receiving the sample and capable of detecting the binding moiety. 
         [0219]    Additionally, according to an embodiment of the present invention, the method further includes the step of calibrating the device by detecting a population of the fluorescently-labeled particles. 
         [0220]    According to another embodiment of the present invention, the particles include the same fluorescent label as the fluorescently-labeled binding moiety. 
         [0221]    Additionally, according to an embodiment of the present invention, the method further includes a second population of particles that include the same fluorescent label as the second fluorescently-labeled binding moiety. 
         [0222]    Moreover, according to an embodiment of the present invention, the method further includes performing an internal calibration after the detecting the fluorescently-labeled binding moiety. 
         [0223]    Notably, according to an embodiment of the present invention, the calibration is completed in less than 5 minutes. 
         [0224]    According to some embodiments, the particles are microbeads. 
         [0225]    Additionally, according to an embodiment of the present invention, the method is performed in less than 15 minutes. 
         [0226]    Furthermore, according to an embodiment of the present invention, the method, further includes the step of determining the presence of at least one cell population in the sample that is not bound by the binding moiety, thus providing an internal negative control for the sample. 
         [0227]    The present invention will be more fully understood from the following detailed description of the preferred embodiments thereof, taken together with the drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0228]    The invention will now be described in connection with certain preferred embodiments with reference to the following illustrative figures so that it may be more fully understood. 
           [0229]    With specific reference now to the figures in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. 
           [0230]    In the drawings: 
           [0231]      FIG. 1  is a simplified schematic illustration showing an apparatus for detecting a biological condition, in accordance with an embodiment of the present invention; 
           [0232]      FIG. 2  is a simplified flow chart of a method for detecting a biological condition, in accordance with an embodiment of the present invention; 
           [0233]      FIG. 3  is a simplified schematic illustration showing a methodology for detecting a biological condition associated with a CD64 cell surface antigen, in accordance with an embodiment of the present invention; 
           [0234]      FIG. 4  is a simplified flow chart of a method for detecting a biological condition associated with a CD64 cell surface antigen, in accordance with an embodiment of the present invention; 
           [0235]      FIG. 5A  is a graphical output of a fluorescent detection assay of a non-activated neutrophil signature associated with the method of  FIGS. 3-4 , in accordance with an embodiment of the present invention; 
           [0236]      FIG. 5B  is a graphical output of a fluorescent detection assay of an activated neutrophil signature, associated with the method of  FIGS. 3-4 , in accordance with an embodiment of the present invention; 
           [0237]      FIG. 5C  is a graphical output of a fluorescent detection assay of a monocyte signature, associated with the method of  FIGS. 3-4 , in accordance with an embodiment of the present invention;  FIG. 5D  is a graphical output of a fluorescent detection assay of a reference bead signature, associated with the method of  FIGS. 3-4 , in accordance with an embodiment of the present invention; 
           [0238]      FIG. 6  is a simplified flow chart of a method for differentiating between different particles, in accordance with an embodiment of the present invention; 
           [0239]      FIG. 7  is a graphical output of fluorescence from reference beads in eight wavebands, in accordance with an embodiment of the present invention; 
           [0240]      FIG. 8  is a graphical output of data from  FIG. 7  after a first mathematical manipulation, in accordance with an embodiment of the present invention; 
           [0241]      FIG. 9  is a graphical output of data from  FIG. 7  after a second mathematical manipulation, in accordance with an embodiment of the present invention; 
           [0242]      FIG. 10  is a graphical output of data from  FIG. 7  after a third mathematical manipulation, in accordance with an embodiment of the present invention; 
           [0243]      FIG. 11  is a graphical output of an event locator, based on data from  FIG. 8-10 , in accordance with an embodiment of the present invention; 
           [0244]      FIG. 12  is a scatterplot matrix of the four fluors signatures showing four distinct event groups, in accordance with an embodiment of the present invention; 
           [0245]      FIG. 13A  is a flowchart of a specific implementation of an algorithm for selecting groups of data from a scatterplot, in accordance with an embodiment of the present invention; 
           [0246]      FIG. 13B  is a flowchart of a general implementation of an algorithm for selecting groups of data from a scatterplot, in accordance with an embodiment of the present invention; 
           [0247]      FIG. 14A  is a histogram of data of Starfire Red (SFR) signature values, in accordance with an embodiment of the present invention; 
           [0248]      FIG. 14B  is a plot of a polynomial and first and second derivative thereof of the histogram shown in  FIG. 14A , in accordance with an embodiment of the present invention; 
           [0249]      FIG. 15A  is a histogram of data of PE488 signature values, in accordance with an embodiment of the present invention; 
           [0250]      FIG. 15B  shows a polynomial fitted to the histogram in  FIG. 15A  as well as corresponding first and second derivatives, in accordance with an embodiment of the present invention; 
           [0251]      FIG. 16A  is a histogram of data of PEAF488 signature values, in accordance with an embodiment of the present invention; 
           [0252]      FIG. 16B  shows a polynomial fitted to the histogram in  FIG. 16A  as well as corresponding first and second derivatives, in accordance with an embodiment of the present invention. 
           [0253]      FIG. 17A  is a histogram of data of Diode 1 channel signature values, in accordance with an embodiment of the present invention; 
           [0254]      FIG. 17B  shows the polynomial fitted to the histogram in  FIG. 17A  as well as the corresponding first and second derivatives, in accordance with an embodiment of the present invention; 
           [0255]      FIG. 18A-18N  is a sequential set of schematic drawings of the operation of an apparatus ( FIG. 1 ) for detecting a biological condition, in accordance with an embodiment of the present invention; and 
           [0256]      FIG. 19  is a simplified three dimensional front view of a system, comprising a reader assembly and a cartridge, for detecting a biological condition, in accordance with an embodiment of the present invention. 
       
    
    
       [0257]    In all the figures similar reference numerals identify similar parts. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0258]    In the detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that these are specific embodiments and that the present invention may be practiced also in different ways that embody the characterizing features of the invention as described and claimed herein. 
         [0259]    International patent application publication no. WO2011/128893 to Kasdan et al., describes a device, system and method for rapid determination of a medical condition and is incorporated herein by reference. 
         [0260]    The microfluidic cartridges of the present invention may be any suitable cartridge as shown in the figures or any of the prior art cartridges described or cited herein, such as, but not limited to, those described in U.S. Pat. No. D669,191 S1, US20120266986 A1, EP1846159 A2, US2012275972, WO11094577A, US2007292941A and EP1263533 B1. 
         [0261]    Reference is now made to  FIG. 1 , which is a simplified schematic illustration of an apparatus  100 , which comprises a cartridge  102  for detecting a biological condition, in accordance with an embodiment of the present invention. 
         [0262]    Apparatus  100  comprises cartridge  102  and a number of chemical/biochemical reactants termed herein, treatment compositions  120 ,  122 ,  124 . The treatment compositions are adapted to react, at least in part, with biological specimen, such as a body specimen, to be introduced to the apparatus. The body specimen may be a bodily fluid such as, but not limited to, blood, serum, plasma, urine, saliva, cerebrospinal fluid (CSF), serous fluid, peritoneal fluid and synovial fluid. Additionally or alternatively, the body specimen may be a solid such as a hair, a tooth part, a bone part or a piece of cartilage. 
         [0263]    Apparatus  100  comprises a specimen receiving element  118 , adapted to transfer the specimen to a sample composition chamber  104 . The sample composition chamber comprises one or more transfer elements  105 , adapted to transfer the specimen from the sample composition chamber to one or more other locations in the cartridge. In the non-limiting example shown in  FIG. 1 , transfer element  105  is a conduit in fluid connection with a treatment chamber  112 . 
         [0264]    Additionally, the cartridge comprises a number of treatment composition chambers  106 ,  108 ,  110 , adapted to respectively house a corresponding number of treatment compositions  120 ,  122 ,  124 . These treatment compositions may be liquid, solid or combinations thereof. Apparatus  100  is typically sold commercially as a system with the treatment compositions disposed therein. In some cases, the apparatus  100  may be adapted for a one-off test and may be disposable. In other cases, the apparatus may be re-used. A re-usable apparatus may be adapted to receive additional external compositions (not shown) or may have a plurality of treatment compositions, wherein only a portion is used for each test. 
         [0265]    The apparatus may be constructed and configured such that the treatment composition comprises proteins attached to a surface, such as to beads. A plurality of beads or other structural elements with proteins attached to their surfaces can be made by any one or more of the following methodologies:—
       simple attachment such as by adsorption via electrostatic or hydrophobic interactions with the surface, entrapment in immobilized polymers, etc.   non-covalent or physical attachment;   covalent bonding of the protein to the bead surface   biological recognition (e.g., biotin/streptavidin).   requires two steps: a first layer is formed by silane chemistry such that the surface presents a reactive group (for example epoxy, amino, thiol, etc.), and a second layer (e.g., the protein to be immobilized or a linker molecule) is covalently attached via the immobilized reactive groups.   covalent attachment to functionalized polymer coatings on the interior of the device or linkage to the free end of a self-assembled monolayer (SAM) on a gold surface.       
 
         [0272]    The reaction type may include any one or more of antigen-antibody binding, sandwich (such as antibody-antigen-antibody), physical entrapment, receptor-ligand, enzyme-substrate, protein-protein, aptamers, covalent bonding or biorecognition. 
         [0273]    Cartridge  102  further comprises at least one transfer element  107 ,  109 ,  111  in fluid communication with each respective of treatment composition chamber, each transfer element also being in fluid communication with treatment chamber  112 . These elements are typically microfluidic channels and may be designed for mixing, such as being tortuous in shape. 
         [0274]    Various methodologies for transferring the contents of the treatment composition chambers and the sample composition chamber via the transfer elements to the treatment chamber may be employed, some of which are known in microfluidics technologies. These include air blowing, suction, vacuuming, mechanical transfer, pumping and the like. 
         [0275]    Cartridge  102  further comprises at least one transfer element  113  in fluid communication with treatment chamber  112  and with an evaluation chamber  114 . 
         [0276]    Optionally, evaluation chamber  114  is further in fluid communication with a transfer element  115 , adapted to remove the contents of the evaluation chamber for disposal outside the cartridge. Alternatively, the evaluation chamber may have no external disposal means. 
         [0277]    Table 1 shows some representative applications of apparatus  100  and methods of the present invention. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Applications of the apparatus and methods of this invention. 
               
             
          
           
               
                   
                   
                   
                   
                 This 
                   
               
               
                   
                   
                   
                 Typical Prior 
                 invention 
               
               
                   
                   
                 Relevant 
                 Art Laboratory 
                 Turn- 
               
               
                   
                   
                 Figures in 
                 Turnaround 
                 around 
               
               
                   
                 Type of 
                 this 
                 time (TAT)- 
                 time 
               
               
                 Application 
                 Test 
                 invention 
                 see references 
                 (TAT) 
                 References 
               
               
                   
               
               
                 Application #1 - 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 U.S. Pat. No. 8,116,984, 
               
               
                 CD64 Infection 
                 Marker 
                 and 3-5D 
                   
                 minutes 
                 Davis, BH et al., 
               
               
                 &amp; Sepsis 
                   
                   
                   
                   
                 (2006) 
               
               
                 1 - Fetal 
                 Plasma 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Dziegiel et al. 
               
               
                 Hemoglobin 
                 Protein 
                 and 6-8D 
                   
                 minutes 
                 (2006) 
               
               
                 Test 
               
               
                 2 - Low Platelet 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Segal, H. C., et al. 
               
               
                 Count 
                 Marker 
                 and 3-5D 
                   
                 minutes 
                 (2005): 
               
               
                 3 - Resolving 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Guerti, K., et al. 
               
               
                 BLAST Flag for 
                 Marker 
                 and 3-5D 
                   
                 minutes 
               
               
                 hematology Lab 
               
               
                 4 - CD34 Stem 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Sutherland et al. 
               
               
                 Cell 
                 Marker 
                 and 3-5D 
                   
                 minutes 
                 (1996) 
               
               
                 Enumeration 
               
               
                 Assay 
               
               
                 5 - Platelets 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Graff et al. (2002) 
               
               
                 Activation 
                 Marker 
                 and 3-5D 
                   
                 minutes 
                 Divers, S. G., et al. 
               
               
                 Assay CD62 
                   
                   
                   
                   
                 (2003) 
               
               
                 6 - D-dimer 
                 Plasma 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Stein et al. (2004) 
               
               
                 (Bead based 
                 Protein 
                 and 6-8D 
                   
                 minutes 
                 Rylatt, D. B., et al. 
               
               
                 protein) 
                   
                   
                   
                   
                 (1983): 
               
               
                 7 -  
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Hillier et al. (1988) 
               
               
                 Chorioamnioitis 
                 Marker 
                 and 3-5D 
                   
                 minutes 
               
               
                 CD64 
               
               
                 8 - CD20 Cell 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Rawstron et al. 
               
               
                 Quantitation 
                 Marker 
                 and 3-5D 
                   
                 minutes 
                 (2001) 
               
               
                 (Therapy 
                   
                   
                   
                   
                 Cheson et al. 
               
               
                 Monitoring 
                   
                   
                   
                   
                 (1996) 
               
               
                 9 - CD52 Cell 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Rawstron et al. 
               
               
                 quantitation 
                 Marker 
                 and 3-5D 
                   
                 minutes 
                 (2001) 
               
               
                 (Therapy 
               
               
                 Monitoring) 
               
               
                 10 - Circulating 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Cristofanilli et al. 
               
               
                 Tumor Cells 
                 Marker 
                 and 3-5D 
                   
                 minutes 
                 (2004 
               
               
                 11 - Reticulated 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Matic et al. (1998) 
               
               
                 Platelet Assay 
                 Marker 
                 and 3-5D 
                   
                 minutes 
                 Ault et al (1993) 
               
               
                   
                   
                   
                   
                   
                 Wang et al. (2002) 
               
               
                 12 - Bacteria 
                   
                   
                 4 hours 
                 10 
                 Blajchman et al 
               
               
                 Detection in 
                   
                   
                   
                 minutes 
                 (2005) 
               
               
                 platelet packs 
                   
                   
                   
                   
                 McDonald et al. 
               
               
                   
                   
                   
                   
                   
                 (2005) 
               
               
                 13 - Platelet 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Michelson (1996) 
               
               
                 Associated 
                 Marker 
                 and 3-5D 
                   
                 minutes 
               
               
                 Antibodies 
               
               
                 14 - Residual 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Bodensteiner, 
               
               
                 Leukocyte 
                 Marker 
                 and 3-5D 
                   
                 minutes 
                 (2003) 
               
               
                 Count in blood 
               
               
                 products 
               
               
                 15 - CD4 HIV 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Rodriguez (2005). 
               
               
                 AIDS 
                 Marker 
                 and 3-SD 
                   
                 minutes 
                 Dieye et al. (2005) 
               
               
                 16 - Leukemia 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Drexler et al (1986) 
               
               
                 Panels - Very 
                 Marker 
                 and 3-5D 
                   
                 minutes 
               
               
                 complex 
               
               
                 17 - Bladder 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Ramakumar et al 
               
               
                 Cancer 
                 Marker 
                 and 3-5D 
                   
                 minutes 
                 (1999) 
               
               
                 Screening in 
                   
                   
                   
                   
                 Lotan et al. (2009) 
               
               
                 Urine - Urine 
               
               
                 sample 
               
               
                 18 - HLA DR 
                 Surface 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Hershman et al. 
               
               
                 Sepsis and 
                 Marker 
                 and 3-5D 
                   
                 minutes 
                 (2005) 
               
               
                 Immunosuppression 
                   
                   
                   
                   
                 Perry et al (2003) 
               
               
                 19 - RECAF 
                 Plasma 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Moro et al. (2005). 
               
               
                 Protein for 
                 Protein 
                 and 6-8D 
                   
                 minutes 
               
               
                 Canine and 
               
               
                 other Cancers 
               
               
                 20 - Cytolmmun - 
                   
                   
                 4 hours 
                 10 
                 Hilfrich et al. 
               
               
                 Cervical 
                   
                   
                   
                 minutes 
                 (2008) 
               
               
                 Screening 
               
               
                 21 - 
                 Plasma 
                 FIGS. 1-2 
                 4 hours 
                 10 
                 Assicot et al. 
               
               
                 Procalcitonin 
                 Protein 
                 and 6-8D 
                   
                 minutes 
                 (1993) 
               
               
                 (Bead Based 
                   
                   
                   
                   
                 Christ-Crain et al. 
               
               
                 Protein) + 
                   
                   
                   
                   
                 (2004) 
               
               
                 Feasibility 
               
               
                   
               
             
          
         
       
     
         [0278]    Reference is now made to  FIG. 2 , which is a simplified flow chart  200  of a method for detecting a biological condition, in accordance with an embodiment of the present invention. 
         [0279]    It should be understood that each of the steps of the method may take a predetermined period of time to perform, and in between these steps there may be incubation and/or waiting steps, which are not shown for the sake of simplicity. 
         [0280]    In a sample transferring step  202 , a sample, such as a bodily specimen is transferred from outside apparatus  100  via receiving element  118  into sample composition chamber  104  and then to the treatment chamber  112 . According to some embodiments, the volume of the specimen or sample is less than 200 μL, less than 100 μL, less than 50 μL, less than 25 μL or less than 11 μL. 
         [0281]    Thereafter, treatment composition  120  is transferred via transfer element  107  to the treatment chamber in a composition transfer step  204 . In some cases, there may be a treatment composition disposed in the treatment chamber. 
         [0282]    Depending on the nature of the treatment composition and sample/specimen type, there may be a requirement to mix or agitate the treatment chamber contents in an optional mixing step  206 . This may be performed by using a small stirbar (not shown) disposed in the chamber. Additionally or alternatively, this may be effected by the fluid dynamics of system. Additionally or alternatively, stirbars may be disposed in any of the other chambers in the apparatus. 
         [0283]    Typically, the total sample volumes are in the range of 10 to 1000 μL, 100 to 900 μL, 200 to 800 μL, 300 to 700 μL, 400 to 600 μL, or 420 to 500 μL. 
         [0284]    According to some embodiments, the volume of the treatment composition chambers  106 ,  108 ,  110  (also called blisters) is from about 1 μL to 1000 μL. According to other embodiments, the volume of the specimen is from about 10 μL to 200 μL. According to other embodiments, the volume of the specimen is about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 μL. 
         [0285]    According to some embodiments, the volume of the treatment compositions  120 ,  122 ,  124  is at most about 500 μL. According to other embodiments, the volume of the specimen is at most about 200 μL. According to other embodiments, the volume of the specimen at most about 500, 450, 400, 350, 300, 250, 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 1 μL. 
         [0286]    According to some embodiments, the volume of a reactant is at least about 1 μL. According to other embodiments, the volume of the specimen is from about 10 μL. According to other embodiments, the volume of the specimen is at least about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 μL. 
         [0287]    The sequence of transfer of the various treatment compositions may be important to the reaction sequence and is typically predefined. Steps  204 - 206  may be performed, for example on treatment composition chamber  106 , thereafter on treatment composition chamber  108  and thereafter on treatment composition chamber  110 . In some cases, some of these steps may be performed concurrently. 
         [0288]    In a checking step  208 , it is ascertained whether all the compositions required for the sample treatment have been transferred to the treatment chamber. If any compositions remain, then steps  204 - 206  are performed on the subsequent treatment composition chamber(s). If no further treatment compositions require transfer, then the sample/specimen is transferred from chamber  104  into the treatment chamber. 
         [0289]    Thereafter, in a second sample transfer step  210 , the sample is transferred from the sample composition chamber into the treatment chamber. 
         [0290]    According to some embodiments, step  210  may be performed before steps  204 - 208 . 
         [0291]    If required, an optional mixing step  212  to the contents of the treatment chamber may be performed. 
         [0292]    In a transferring step  214 , the contents of the treatment chamber are transferred to the evaluation chamber. 
         [0293]    The evaluation chamber  114  is configured and constructed for one or more evaluation steps  216 . These may include any of the following, or combinations thereof:
       a) transfer of radiation there-through,   b) impinging radiation thereupon;   c) detecting reflected, refracted, and/or transmitted radiation,   d) detecting emitted radiation;   e) capturing one or more images thereof;   f) performing image analysis on the captured images;   g) measuring electrical characteristics of the treated specimen;   h) impinging sonic energy thereon;   i) detecting sonic energy therefrom; and   j) analyzing the outputs of any one or more of the above steps.       
 
         [0304]    According to some embodiments, the cartridge is introduced into a system as described in International patent application publication no. WO2011/128893 to Kasdan et al., incorporated herein by reference. 
         [0305]    The results of the evaluation step are then outputted in a results outputting step  218 . 
         [0306]    According to some embodiments; the apparatus may have on-board means for showing a result, such as a colorimetric strip (not shown). Additionally or alternatively, the results are displayed in a display unit, separate and remote from apparatus  100 . 
         [0307]    Reference is now made to  FIG. 3 , which is a simplified schematic illustration showing a methodology  300  for detecting a biological condition associated with a CD64 cell surface antigen, in accordance with an embodiment of the present invention. 
         [0308]    According to some embodiments, the method is carried out in the apparatus shown in  FIG. 1  and as described herein. A biological specimen, such as a blood sample, is aspirated via specimen receiving element  118  to sample composition chamber  104 , and then to treatment chamber  112 . The sample is typically of a volume in the range of 10-200 μL. 
         [0309]    The blood sample is typically whole blood recently removed from a patient. The whole blood comprises mainly red blood cells (also called RBCs or erythrocytes), platelets and white blood cells (also called leukocytes), including lymphocytes and neutrophils. Increased number of neutrophils, especially activated neutrophils are normally found in the blood stream during the beginning (acute) phase of inflammation, particularly as a result of bacterial infection, environmental exposure and some cancers. 
         [0310]    A cocktail  304  comprising antibodies to CD64 and antibodies to CD163 is introduced to the treatment chamber (see Davis et al. (2006)). Each antibody type is typically tagged by a specific fluorescent tag. 
         [0311]    The contents of the chamber are incubated and/or mixed as is required to bind the activated blood neutrophils with the CD64 tagged antibody (also called a marker) to form activated neutrophils with CD64 marker  310 , and/or monocyte with a CD64 tagged antibody and a CD163 tagged antibody  312 . Lymphocytes with no markers  314  are present in the contents, as well as unaffected RBCs  316 . 
         [0312]    Thereafter, a lysis reagent or diluent  306  is introduced into treatment chamber  112 . In the case of a lysis reagent, it is adapted to lyse red blood cells to form lysed red blood cells  324 . Additionally, reference/calibration beads  308  are added to the treatment chamber. These are used to calibrate the outputs, as is explained with reference to  FIGS. 5A-5D  hereinbelow. 
         [0313]    CD64 (Cluster of Differentiation 64) is a type of integral membrane glycoprotein known as an Fc receptor that binds monomeric IgG-type antibodies with high affinity. Neutrophil CD64 expression quantification provides improved diagnostic detection of infection/sepsis compared with the standard diagnostic tests used in current medical practice. 
         [0314]    CD163 (Cluster of Differentiation 163) is a human protein encoded by the CD163 gene. It has also been shown to mark cells of monocyte/macrophage lineage. 
         [0315]    Reference is now made to  FIG. 4 , which is a simplified flow chart  400  of a method for detecting a biological condition associated with a CD64 cell surface antigen, in accordance with an embodiment of the present invention. 
         [0316]    According to some embodiments, the method is carried out in the apparatus shown in  FIG. 1  and as described herein. In a first transferring step  402 , a biological specimen, such as a blood sample is aspirated via specimen receiving element  118  to sample composition chamber  104 . The sample is typically of a volume in the range of 10-200 μL. 
         [0317]    Typically, the total sample volumes are in the range of 10 to 1000 μL, 100 to 900 μL, 200 to 800 μL, 300 to 700 μL, 400 to 600 μL, or 420 to 500 μL. 
         [0318]    According to some embodiments, the volume of the treatment composition chambers  106 ,  108 ,  110  (also called blisters) is from about 1 μL to 1000 μL. According to other embodiments, the volume of the specimen is from about 10 μL to 200 μL. According to other embodiments, the volume of the specimen is about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 μL. 
         [0319]    According to some embodiments, the volume of the treatment compositions  120 ,  122 ,  124  is at most about 500 μL. According to other embodiments, the volume of the specimen is at most about 200 μL. According to other embodiments, the volume of the specimen at most about 500, 450, 400, 350, 300, 250, 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 1 μL. 
         [0320]    According to some embodiments, the volume of a reactant is at least about 1 μL. According to other embodiments, the volume of the specimen is from about 10 μL. According to other embodiments, the volume of the specimen is at least about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 μL. 
         [0321]    In an addition step  404 , a cocktail of tagged antibodies to CD64 and to CD163 is added to the treatment chamber  112  and is incubated with the blood sample. In the incubation phase of this step, the antibodies bind activated neutrophils with CD64 marker  310 , and/or monocytes activated with a CD64 tagged antibody and a CD163 tagged antibody  312 . 
         [0322]    In a lysis reagent addition step  406 , the lysis reagent is added to the treatment chamber and thereby lyses at least some of the RBCs in the chamber. 
         [0323]    At any suitable time, typically following lysis step  406 , reference beads are added to the contents of the treatment chamber in a reference bead adding step  408 . 
         [0324]    After a predefined period of time, an analysis step  410  is performed to analyze the fluorescent emission signatures from the contents. This is described in further detail with reference to  FIGS. 5A-5D . According to some examples, the evaluation chamber  114  is constructed and configured to allow cells to pass through a reading zone  130  such that each cell passing therethrough is analyzed individually. The assay sensitivity is around 86% and its specificity is around 87% (Hoffmann, 2011). 
         [0325]    The time required to complete an assay using apparatus  100  of the present invention varies depending on a number of factors, with non-limiting examples that include described herein. In some embodiments, the time required to complete an assay is from about 0.5 to 100 minutes. In other embodiments, the time required to complete an assay is from about 1 to 20 minutes. In still other embodiments, the time required to complete an assay is from about 1 to 10 minutes. In some examples, the time required to complete an assay is from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 80, or 100 minutes. 
         [0326]    Reference is now made to  FIG. 5A , which is a graphical output of a fluorescent detection assay of a non-activated neutrophil signature  500  associated with the method of  FIGS. 3-4 , in accordance with an embodiment of the present invention. The non-activated tagged neutrophils each emit a signal  502  at wavelength W 1  of an intensity I 1 . The wavelengths shown in  FIGS. 5A-5D  represent a peak wavelength of waveband outputs detected, as are shown in  FIGS. 7-11 . 
         [0327]      FIG. 5B  shows a graphical output of a fluorescent detection assay of an activated neutrophil signature  510 , associated with the method of  FIGS. 3-4 , in accordance with an embodiment of the present invention. Each activated tagged neutrophil emits an activated neutrophil signature  512  at wavelength W 1  of an intensity I 2 . Typically I 2  is greater than I 1 . In some cases the difference in signatures  512  and  510  may be detected by an image analysis, a fluorescent emission radiation count or by other qualitative or quantitative methods known in the art. The current example is not meant to be limiting. 
         [0328]    Turning to  FIG. 5C , there can be seen a graphical output of a fluorescent detection assay of a monocyte signature  520 , associated with the method of  FIGS. 3-4 , in accordance with an embodiment of the present invention. The monocyte signature comprises a first signal  522  at a first wavelength W 1  of an intensity I 3  and a second signal  524  at a second wavelength W 2  of an intensity I 4 . 
         [0329]      FIG. 5D  shows a graphical output of a fluorescent detection assay of a reference bead signature  530 , associated with the method of  FIGS. 3-4 , in accordance with an embodiment of the present invention. The reference bead signature comprises a first signal  532  at a first wavelength W 1  of an intensity I 1  (similar or equal to non-activated tagged neutrophils&#39; signal  502 ) and a second signal  534  at a second wavelength W 3  of an intensity I 5 . 
         [0330]    This methodology enables the identification and quantification of activated neutrophils by intensity of signature  512  of the CD64 tag. Monocytes are identified by the double signal signature  522 ,  524 , acting as a positive control. Reference beads are identified by the unique signal  534  at wavelength W 3 . The intensity of signal  532  at wavelength W 1  provides a reference level of CD64 for the comparison of intensity of  512  of the neutrophils. 
         [0331]    Lymphocytes with no markers  330  ( FIG. 3 ) act as a negative control and should provide no fluor signature, but may be detected by their scattering or other characteristics. Further details of some embodiment of this assay procedure are described in U.S. Pat. No. 8,116,984 and in Davis, B H et al., (2006). 
         [0332]    Reference is now made to  FIG. 6 , which is a simplified flow chart of a method  600  for differentiating between different particles, in accordance with an embodiment of the present invention. 
         [0333]    The input to the processing is a time series from each of the channels in the eight channel photomultiplier array  601 . In addition, data from multiple scatter channels  609  is introduced. Each fluorescent time series and scatter time series may be processed individually employing respective spectral cross-correlation algorithm  606  and scatter algorithm  607  to smooth it and minimize noise. Two possible processing methods are boxcar averaging algorithm  602  and matched filtering algorithm  604 . In addition, groups of individual channels may be correlated to yield a multiple spectral cross-correlations  606 . One or more of these derived time series may be used to determine event locations. 
         [0334]    Once an event is located in the eight channel time series the composition of that event in terms of known fluorophore signatures is determined using a minimum mean square error fit  610 . The event is now described in terms of its composition of known fluors. Each event thus described is stored in an event store, i.e. memory, together with the data from the eight time series for that event and its description  612 . Based on the fluor composition for each event in the data store, it is possible to determine the type of particle. For example, a neutrophil  616  is characterized by the single fluor attached to the CD64 antibody shown in  FIG. 5  as W 1 . Thus events that are preponderantly characterized by the single fluor attached to the CD64 antibody are identified as neutrophils. 
         [0335]    Similarly, monocytes  618  are characterized by fluors W 1  and W 2  so that an event with both of these fluor signatures is identified as a monocyte. Similarly, a bead  620  is characterized by an event that has fluors W 1  and W 3 . Lymphocytes  622  do not express significant fluorescence but are identified by their scatter as events. Events that do not match any of the known combinations of the fluorophores are identified as rejects  626 . 
         [0336]    Given the population of identified events, the median intensity of the neutrophil population and the median intensity of the bead population are determined. The ratio of the neutrophil median to the bead median is the desired Leuko64 index. The positive control value is determined as the median intensity of the CD64 fluorophore bound to monocytes divided by the median intensity of the same fluorophore on the bead population. The negative control value is determined by the median intensity of the CD64 fluorophore bound to lymphocytes. These are the key steps in performing the Leuko64 assay. 
         [0337]      FIG. 7  is a graphical output  700  of fluorescence from reference beads in eight wavebands, in accordance with an embodiment of the present invention. This figure shows the smoothed signals from the eight channel PMT array for two reference beads. The amplitude for each waveband is shown on the same graph. The corresponding wavelength range is shown for each plot  702 ,  706 ,  708 ,  710 ,  712 ,  714 ,  716 ,  718  in the legend box. The two fluorophores signatures present in this plot are  702 , 706  and  708  for FITC, which is the fluorophore attached to the CD64 antibody and  710 ,  712  for Starfire Red, which is the fluorophore identifying the reference beads. 
         [0338]    Reference is now made to  FIG. 8 , which is a graphical output  800  of data from  FIG. 7  after a first mathematical manipulation, in accordance with an embodiment of the present invention.  FIG. 8  shows the cross correlation of wave bands one two and three corresponding to wavelength 500 to 525, 525 to 550, and 552 to 575 nm. This cross correlation is computed by multiplying the boxcar smoothed time series corresponding to these wavelengths. This signal will have a high-value when an event containing the FITC fluorophore is present. 
         [0339]      FIG. 9  is a graphical output  900  of data from  FIG. 7  after a second mathematical manipulation, in accordance with an embodiment of the present invention.  FIG. 9  shows the cross correlation of wave bands 3, 4 and 5 corresponding to wavelengths 550 to 575, 575 to 600, and 600 to 625 nm. This signal will have a high-value when an event containing the PE fluorophore is present. 
         [0340]      FIG. 10  is a graphical output  1000  of data from  FIG. 7  after a third mathematical manipulation, in accordance with an embodiment of the present invention.  FIG. 10  shows the cross correlation of wave bands 7 and 8 corresponding to wavelengths 650 to 675, and 675 to 700 nm. This signal will have a high-value when an event containing the Starfire Red fluorophore is present. 
         [0341]      FIG. 11  is a graphical output  1100  of an event locator, based on data from  FIG. 8-10 , in accordance with an embodiment of the present invention.  FIG. 11  shows the event locations determined from the cross correlations computed in  FIGS. 8 ,  9  and  10 . The solid fill area  1102  corresponds to the region where any of the cross correlations  802 ,  902  and  1002  exceeded a predefined threshold. Similarly, the solid fill area  1104  corresponds to the region where any of the cross correlations  804 ,  904  and  1004  exceeded a predefined threshold. This then completes the event location process. 
         [0342]    Reference is now made to  FIG. 12 , which is a scatterplot matrix  1200  of four fluor signatures  1210 ,  1220 ,  1230  and  1240 , showing four distinct event groups, in accordance with an embodiment of the present invention. 
         [0343]    Fluor signature  1210  is the group of reference beads that contain the reference level of the F488 fluorophore (F488 is the designation of a fluorophore with an emission spectrum identical to or equivalent to Fluorescein isothiocyanate (FITC)) and are identified by their Starfire Red™ (Starfire Red is the designation of a fluorophore or its emission spectrum, produced by Bangs Laboratory, 9025 Technology Dr. Fishers, Ind. 46038-2886, USA) embedded fluorophore. Fluor signature  1220  is the group of monocytes tagged with the phycoerythrin (PE) fluorophore identified by the signature name PE488 (exited by a laser at 488 nm). (PE488 is the designation of a fluorophore with an emission spectrum identical to or equivalent to phycoerythrin (PE)). 
         [0344]    Fluor signature  1230  is the group of lymphocytes tagged with the PEAF488 (Alexa Fluor®610, Life Technologies Corporation, 3175 Staley Rd. Grand Island, N.Y. 14072 USA) fluorophore. Finally, fluor signature  1240  is the group of neutrophils tagged with the F488 (Alexa Fluor® 488 Life Technologies Corporation, 3175 Staley Rd. Grand Island, N.Y. 14072 USA) fluorophore, which is the primary target group for analysis. 
         [0345]    Reference is now made to  FIG. 13A , which is a flowchart  1300  of a specific implementation of an algorithm for selecting groups of data from a scatterplot, in accordance with an embodiment of the present invention. 
         [0346]    The algorithm in  FIG. 13A  is a specific implementation of the general algorithm in  FIG. 13B  to select each of the groups  1210 ,  1220 ,  1230  and  1240  ( FIG. 12 ) and determine specific parameter values in each of the groups. 
         [0347]    In a first ordering signature step  1304  the Star Fire Red (SFR) signature is used to order (from smallest SFR signature to largest) the entire dataset of waveband and signature values  1302 . 
         [0348]    In a second step  1320 , an analysis of a histogram of an SFR signature values as shown in  FIG. 14A  to select the group  1210 . This is a small group  1404  at the upper end of group  1402  in the histogram  1400  in  FIG. 14A . The next step is to remove this group from the overall dataset as shown in  FIG. 13  Step  1322 . The removed group is the bead dataset  1324 . 
         [0349]    A dataset of Waveband and Signature values with bead dataset removed  1340  is then manipulated as follows. In an ordering step  1342 , the data is organized according to its PE (phycoerythrin) signature from smallest to largest PE (phycoerythrin) signature. 
         [0350]    In an analyzing PE histogram set step,  1344 , the data is manipulated to find a group corresponding to monocytes. 
         [0351]    In an extracting monocytes dataset step  1346 , a monocyte dataset of waveband and signature values  1348  is extracted. A dataset of waveband and signature values with beads and monocytes removed  1360  is then further processed as follows. Set  1360  is organized according to its PEAF (PEAF488) (see above for beads and PE) signature in an order according to PEAF signature ordering step  1362 . 
         [0352]    In an analyzing PEAF histogram to find a group corresponding to lymphocytes step  1364 , set  1360  is analyzed to determine if any of the data have behavior corresponding to lymphocytes. 
         [0353]    In an extraction step  1366 , a lymphocyte dataset of waveband and signature values  1368  is extracted from set  1360  and the remaining dataset is a dataset of waveband and signature values with bead, monocytes and lymphocytes removed  1380 . 
         [0354]    In an order by Diodel signature step  1382 , dataset  1380  is analyzed according to a Diodel signature (see above). Dataset  1380  is then analyzed in an analyzing step  1384  to find a group of data having properties of neutrophils. 
         [0355]    In an extracting step  1386 , a group of data having properties of non-neutrophils  1388  is removed. A remaining group  1391  (assumed to be neutrophils) is used in a computing step  1392  to compute desired metric from the group parameters. 
         [0356]    Reference is now made to  FIG. 13B , which is a flowchart of a general implementation of an algorithm  1350  for selecting groups of data from a scatterplot, in accordance with an embodiment of the present invention. 
         [0357]    In a first ordering signature step  1305  a first signature is used to order the dataset of waveband and signature values  1303 . 
         [0358]    In a second step  1321 , an analysis of a histogram of a 1st signature values to find the group corresponding to 1 st  signature  1325 , as exemplified in  FIG. 14A  to select the group  1210 . This is a small group  1404  at the upper end of group  1402  in the histogram  1400  in  FIG. 14A . It should be noted that this is but one way to select the group and other methods employing additional data set values in combination may be used. The next step is to remove this group from the overall dataset as shown in  FIG. 13B  Step  1323 . A removed group is a 1st signature dataset  1325 . 
         [0359]    A dataset of Waveband and Signature values with 1st dataset removed  1341  is then manipulated as follows. In an ordering step  1343 , the data is organized according to its 2nd signature. 
         [0360]    In an analyzing 2 nd  signature histogram set step,  1345 , the data is manipulated to find a group corresponding to the 2 nd  signature. 
         [0361]    In an extracting 2 nd  signature dataset step  1347 , a 2 nd  signature dataset of waveband and signature values  1349  is extracted. A dataset of waveband and signature values with 1 st  and 2 nd  signatures groups removed  1361  is then further processed as follows. Set  1361  is organized according to its i th  signature in an order according to i th  signature ordering step  1363 . 
         [0362]    In an analyzing i th  histogram to find a group corresponding to i th  signature step  1365 , set  1361  is analyzed to determine if any of the data have behavior corresponding to the i th  signature. 
         [0363]    In an i th  signature extraction step  1367 , an i th  signature dataset of waveband and signature values  1369  is extracted from set  1381  and the remaining dataset is a dataset of waveband and signature values with 1 st  2 nd  and i th  signature groups removed  1381 . 
         [0364]    In an order by N th  signature step  1383 , dataset  1381  is analyzed according to an N th  signature. Dataset  1381  is then analyzed in an analyzing step  1385  to find a group of data having properties of not having Nth signature properties. 
         [0365]    In an extracting step  1387 , a group of data having properties of non-Nth signatures  1397  is removed. A remaining group  1395  (assumed to be Nth groups) is used in a computing step  1393  to compute desired metric from the group parameters. 
         [0366]      FIG. 14A  is a histogram  1400  of data of Starfire Red (SFR) signature values, in accordance with an embodiment of the present invention. 
         [0367]      FIG. 14B  shows a plot  1450  of a polynomial  1452  and first derivative thereof  1456  and second derivative thereof  1458  of histogram  1400  shown in  FIG. 14A , in accordance with an embodiment of the present invention. 
         [0368]    Referring to  FIG. 14B , the method of determining an upper group  1404  in  FIG. 14A  is as follows. A polynomial  1452  of sufficient degree is fitted to the histogram data  1454  (as shown in  FIG. 13A , set  1324 ) is shown in  FIG. 14B . The first derivative  1456  and the second derivative  1458  of this polynomial are computed. A plurality of zeros  1460  of the first derivative are indicated by the square boxes along the zero line. A point where the polynomial is both maximum and has a zero derivative  1462  is indicated by the box with an X in it. This point in the histogram corresponds to the peak of the large group  1402  (FIG.  1402 ). A next zero  1464  of the derivative of the polynomial corresponds to the end of the large group in the histogram. All points in the histogram above this value are in the small group. Since the dataset has been ordered from smallest to largest based on the value of SFR488, and the histogram horizontal axis is also ordered from smallest to largest value of SFR488 the point at which the large group ends is the value of SFR488 above which records in the SFR488 ordered dataset are to be removed and identified as the bead dataset  1324  of waveband and signature values as indicated in  FIG. 13A . 
         [0369]      FIG. 15A  is a histogram  1500  of data of PE488 signature values, in accordance with an embodiment of the present invention. 
         [0370]      FIG. 15B  shows a polynomial fitted to the histogram in  FIG. 15A  as well as a corresponding first derivative  1556  and a second derivative  1558 , in accordance with an embodiment of the present invention. 
         [0371]    The records remaining in the dataset are now reordered using the PE488 signature from smallest to largest. Histogram  1500  of the PE488 signature values  1502  is shown in  FIG. 15A . Again in this case, there is a small group  1504  to the right of the large group  1502  which corresponds to the desired monocyte population.  FIG. 15B  shows the polynomial  1552  fitted to data  1554  of histogram  1500  in  FIG. 15A  as well as the corresponding first and second derivatives. The upper group  1504  is determined in the same way as the upper group of the SFR488 histogram as was described previously. It should be noted that while in both of these cases only a one dimensional histogram was analyzed and used as the basis for selecting the desired population, multiple fields from each record in the dataset may be used to effect a group selection. As noted in  FIG. 15 , the monocyte group  1504  is removed from the dataset which now contains primarily lymphocytes, neutrophils and other particles such as un-lysed erythrocytes and other debris. 
         [0372]      FIG. 16A  is a histogram  1600  of data  1602  of PEAF488 signature values, in accordance with an embodiment of the present invention. 
         [0373]      FIG. 16B  shows a polynomial  1652  fitted to histogram data  1654  from  FIG. 16A  as well as a corresponding first derivative  1656  and a second derivative  1658 , in accordance with an embodiment of the present invention. 
         [0374]    The records remaining in the dataset are now reordered using a PEAF488 signature corresponding to lymphocytes. A histogram  1600  of the PEAF488 signature is shown in  FIG. 16A  and the corresponding polynomial fit with its first and second derivatives are shown in  FIG. 16B . The process outlined above is applied in this case as well to identify and remove a small group  1604  appearing at an upper end of the histogram, from a large group  1602 . The lymphocyte group is now removed as shown in  FIG. 13A  leaving a dataset  1380  which now contains primarily neutrophils and other particles such as unlysed erythrocytes and other debris. 
         [0375]    While neutrophils  1391  are tagged with a fluorophore with an F488 signature, other particles appear to express this signature because of the unbound fluorophore in solution. The other particles, however, are smaller than neutrophils, which now comprise the group with the largest forward scatter as measured by a Diodel (forward scatter detector) channel. A histogram of the Diodel channel is shown in  FIG. 17A . 
         [0376]      FIG. 17A  is a histogram  1700  of data of Diode 1 channel signature values, in accordance with an embodiment of the present invention. 
         [0377]      FIG. 17B  shows a polynomial  1752  fitted to data  1754  from the histogram in  FIG. 17A , as well as a corresponding first derivative  1756  and a second derivative  1758 , in accordance with an embodiment of the present invention. 
         [0378]    As described above, an upper group  1704  ( FIG. 17A ) corresponding to larger particles, which are the neutrophils is selected. This completes the decomposition of the original dataset  1302  into the four distinct event groups ( 1324 ,  1348 ,  1368 ,  1391 ) shown in  FIG. 13A . 
         [0379]    Within each group various parameters may be computed from the fields in the dataset. An example is shown in the following table. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Observa- 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 tions 
                 NAM 
                 MEDUG 
                 MEDF488 
                 MEDWaveband2 
                 MEDWaveband2N 
                 INDEX488 
                 INDEXWaveband2 
                 INDEXWaveband2N 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 SFR488 
                 166 
                 978.72 
                 3395.26 
                 3062.00 
                 503.80 
                 1.00 
                 1.00 
                 1.00 
               
               
                 PE488 
                 73 
                 3851.88 
                 5968.83 
                 5843.50 
                 723.66 
                 1.76 
                 1.91 
                 1.44 
               
               
                 PEAF488 
                 332 
                 1164.38 
                 −4.36 
                 37.00 
                 4.63 
                 0.00 
                 0.01 
                 0.01 
               
               
                 F488 
                 620 
                 379.98 
                 379.98 
                 361.00 
                 37.92 
                 0.11 
                 0.12 
                 0.08 
               
               
                 Diode1 
                 59 
                 7027.00 
                 −113.54 
                 −73.00 
                 −6.81 
                 −0.03 
                 −0.02 
                 −0.01 
               
               
                   
               
             
          
         
       
     
         [0380]    The observations column contains the name of the group. The NAM column is the number of events in the group. The MEDUG column is the median value of the signature for that group. For example in the SFR488 row the median SFR488 signature value is 978.72. The MEDF488 column contains the median value of the F488 signature for the specified group. The MEDWaveband2 column contains the median value of the Waveband2 values in the group. The MEDWaveband2N column contains the median value of the Waveband2N values in the group. The INDEX488 column contains the ratio of the MEDF488 value for the group to that of the SFR488 group. Similarly, INDEXWaveband2 and INDEXWaveband2N are the ratios of the Waveband2 and Waveband2N medians for the group to that of the SFR488 group. 
         [0381]    Although, specific groups corresponding to leukocyte subsets and a specific algorithm to compute a specific index based on these groups has been illustrated, one skilled in the art can use this basic approach whenever it is necessary to select groups from a dataset and compute numeric values based on parameters associated with these groups as shown in the general diagram of figure X. 
         [0382]      FIG. 18A-18N  is a sequential set of schematic drawings of the operation of an apparatus  100  ( FIG. 1 ) for detecting a biological condition, in accordance with an embodiment of the present invention. 
         [0383]    In  FIG. 18A , a blood sample  1801  enters a specimen receiving element  1818  and fills a chamber  1804 . In  FIG. 18B , a blister  1820  comprising a treatment composition  120  ( FIG. 1 ) is pressed and antibody cocktail is mixed with 10 microliters of the blood sample. 
         [0384]    In  FIG. 18C , a mixing bellows  1815  is pressed and this effects mixing of the antibody cocktail and the 10 microliters of the blood sample in a first mixing chamber  1812  to form a first mixture  1803 . 
         [0385]    In  FIG. 18D , the bellows is released and mixture  1803  is siphoned along a tortuous channel  1813  and into a second mixing chamber  1811 . Upon release of the bellows, the first mixture returns from the second mixing chamber, back along the tortuous channel to the first mixing chamber. Every time the bellows is pressed the mixture moves towards the second chamber and every time it is released, it returns, wholly or in part to the first chamber. This mixing may be performed multiple times. 
         [0386]    In  FIGS. 18E-18G , a second composition blister  1822  is pressed, releasing a second composition  122  ( FIG. 1 ), such as a lysis composition thereby forming a second mixture  1805 . The second mixture is mixed by pressing of bellows  1815 , the second mixture returns from the second mixing chamber, back along tortuous channel  1813  to the first mixing chamber. Every time the bellows is pressed the mixture moves towards the second chamber  1811  and every time it is released, it returns, wholly or in part to the first chamber  1812 . This mixing may be performed multiple times. 
         [0387]    In  FIGS. 18H-18J , a third blister  1824  is released comprising a third composition  124  ( FIG. 1 ), such as a control reference, into the second mixing chamber, thereby forming a third composition  1807 . The third mixture is mixed by pressing of bellows  1815 , the third mixture returns from the second mixing chamber, back along tortuous channel  1813  to the first mixing chamber. Every time the bellows is pressed the mixture moves towards the second chamber  1811  and every time it is released, it returns, wholly or in part to the first chamber  1812 . This mixing may be performed multiple times. 
         [0388]    In  FIGS. 18J-18L , a reading bellows  1817  is pressed, which forces some of the third composition towards a reading cuvette  1830 . 
         [0389]    In  FIGS. 18M-18N , particles  1860  from the third composition flow from the cuvette  1830  along a channel  1852  to a reading region  1850 . The cells pass through the reading region and are excited by one or more lasers  1862 ,  1863 . At least one excitation laser beam  1864  impinges on cell  1860  and an emission beam  1866  is detected by a detector  1870 . In one example, this is cell emission fluorescence and detector  1870  is a spectrometer. 
         [0390]    Reference is now made to  FIG. 19 , which is a simplified three dimensional front view of a system  1900  comprising a reader assembly  1901  and a cartridge  1911  for detecting a biological condition, in accordance with an embodiment of the present invention. 
         [0391]    Shown in  FIG. 19  are outer view of the reader assembly  1901  and an inner view  1902  and cartridge  1911 . The cartridge is inserted in the reader assembly as shown. Once the cartridge is inserted in the reader assembly all assay pre-analytical processing and analysis are performed automatically. Results of the analysis are displayed on a user interface touch-screen  1915 , which is also used to control operation of the reader. 
         [0392]    The internal components of the reader assembly are seen in view  1902 ,  FIG. 19 . Reader assembly  1901  comprises an ITX computer,  1922 , a Galil motor controller,  1924 , an electronics power supply  126 , cartridge,  1911 , inserted into a cartridge handling unit (CHU)  1928  and a forward scatter detector  1930 . Not seen are the reader optics  1942 , a data acquisition board  1944  and a general electronics printed circuit board  1946 . 
       Example 
     Application No. 1—CD64 Infection &amp; Sepsis 
       [0393]    A cartridge  102  ( FIG. 1 ) is prepared for receiving a blood sample. The cartridge comprises a number of treatment composition chambers  106 ,  108 ,  110 , adapted to respectively house a corresponding number of treatment compositions  120 ,  122 ,  124 . These compositions are described in further detail in U.S. Pat. No. 8,116,984 and in Davis, B H et al., (2006)), incorporated herein by reference. In brief, Reagent A comprises a mixture of murine monoclonal antibodies (contains buffered saline), Reagent B—10× Concentrated Trillium Lyse solution (contains ammonium chloride), Reagent C—suspension of 5.2 μm polystyrene beads labeled with Starfire Red and fluorescein isothiocyanate (FITC), (contains &lt;0.1% sodium azide and 0.01% Tween 20). 
         [0394]    In a sample transferring step  202  ( FIG. 2 ), a 10 uL blood sample, is transferred from outside apparatus  100  via receiving element  118  into sample composition chamber  104  and then on to treatment chamber  112  in a transferring step  214 . 
         [0395]    An antibody composition (Reagent A)  120  comprising CD64 antibodies is transferred via transfer element  107  to the treatment chamber  112  in a composition transfer step  204 . 
         [0396]    These two steps combined with mixing step  206  take around four minutes using cartridge  102  of the present invention. 
         [0397]    A lysis buffer (Reagent B)  122  is also added and mixed with the resultant mixed composition. This step and mixing all the compositions takes around three minutes using cartridge  102  of the present invention. Reference beads (Reagent C)  308  are added to the treatment chamber. 
         [0398]    The evaluation chamber  114  is configured and constructed for one or more evaluation steps  216 . 
         [0399]    According to some embodiments, the cartridge is introduced into a system as described in International patent application publication no. WO2011/128893 to Kasdan et al., incorporated herein by reference. This system has software associated therewith for computing the CD64 and CD163 indices on leukocytes. 
         [0400]    The results of the evaluation step are then outputted in a results outputting step  218 . According to this example, the time taken from the introduction of the small blood sample to obtaining an indication of sepsis is less than 15 minutes, typically around 10 minutes. 
         [0401]    From a user point of view, the following steps are performed:
       1) The user adds drop of blood to the cartridge  102  and seals it. (10 μL are metered out by microfluidics).   2) Blister A ( 106 ) is pressed, releasing 100 μL of Reagent A. Mixing in the cartridge is controlled by the cartridge handling unit (CHU), followed by a 4-minutes incubation.   3) Blister B ( 108 ) is pressed, releasing ˜250 μL of Reagent B. Mixing in the cartridge is controlled by the CHU, followed by a 3-5-minutes incubation.   4) Magnetic stirbar is activated, stirring the bead suspension (Reagent C).   5) Blister C ( 110 ) is pressed, releasing 100 μL of Reagent C. Mixing in the cartridge is controlled by the CHU. According to one example, Reagent A is a mixture of murine monoclonal antibodies-diluted 1:5 in buffered saline (PBS+0.5% BSA); Reagent B is a Trillium Lyse solution (at working concentration); Reagent C is a suspension of 5.2 μm polystyrene beads labeled with Starfire Red and FITC, diluted 1:100 in PBS+0.01% Tween 20.   6) The sample is read by the optoelectronics core, and collected to the reading below.   7) Data is analyzed automatically and result is presented.   8) The cartridge is disposed as biohazard.       
 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Comparison of Prior art methodology with the methodology 
               
               
                 of the present invention for detecting sepsis using CD64 
               
               
                 and CD163 antibodies. 
               
               
                 LeukoDx device-present invention 
               
             
          
           
               
                   
                   
                 Volume 
                   
                   
               
               
                 Step 
                 Description 
                 (uL) 
                 Duration (min) 
                 comments 
               
               
                   
               
               
                 1 
                 Mixing blood and 
                 Blood- 
                   
                   
               
               
                   
                 antibodies 
                 10 
               
               
                   
                   
                 Abs- 
               
               
                   
                   
                 50 
               
               
                 2 
                 Adding RBC lysis 
                 250 
                 3 
                 Might require 
               
               
                   
                 buffer 
                   
                   
                 heating the buffer 
               
               
                   
                   
                   
                   
                 to 37 C. 
               
               
                 3 
                 Incubating, 
                   
                 3 
               
               
                   
                 Vortexing 
               
               
                 4 
                 Adding 
                  2 
                 Less than 1 
               
               
                   
                 normalization beads 
               
               
                 5 
                 Reading 
                   
                 Less than 1 
               
               
                   
                 Total 
                 312 
                 10  
               
               
                   
               
             
          
         
       
     
         [0410]    In the case of sepsis, by “normalization” is meant taking the ratio of the median of the target population fluorescence emission to the median of the reference bead population fluorescence emission. 
         [0411]    According to some embodiments, the readout may comprise an optoelectronics core, which enables identification and detection of fluorescent signals. The CCD in the core, used for focusing, can also be used to read chemiluminescent signals. The readout to user may also indicate where the result falls relative to reference ranges. 
         [0412]    The contents of these publications are incorporated by reference herein where appropriate for teachings of additional or alternative details, features and/or technical background. It is to be understood that the invention is not limited in its application to the details set forth in the description contained herein or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Those skilled in the art will readily appreciate that various modifications and changes can be applied to the embodiments of the invention as hereinbefore described without departing from its scope, defined in and by the appended claims. 
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