PATENT ABSTRACT
The present invention provides kits, apparatus and methods for determining a biological condition in a mammalian subject, the method includes incubating a specimen from a patient with at least one composition in a kit for a predetermined period of time to form at least one reaction product, when the subject has said biological condition, and receiving an indication of the at least one reaction product responsive to at least one reporter element in the kit thereby providing the indication of the biological condition in the subject.

PATENT DESCRIPTION
CROSS-REFERENCE 
       [0001]    This application is a continuation application of application Ser. No. 14/296,317, filed on Jun. 4, 2014, which is a divisional application of Ser. No. 13/716,246, filed Dec. 17, 2012, which is incorporated herein by reference in its entirety. 
         [0002]    The disclosures of the co-pending US Provisional Patent Application to Kasdan, et al, filed on Nov. 17, 2012, and titled “Kits, Compositions and Methods for Detecting a Biological Condition” and the co-pending US Provisional Patent Application to Kasdan, et al, filed on Nov. 17, 2012, and titled “Kits, Compositions and Methods for Rapid Chemical Detection” are incorporated herein by reference in their entirety. 
     
    
     FIELD OF THE INVENTION 
       [0003]    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 
       [0004]    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. 
         [0005]    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. 
         [0006]    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 
       [0007]    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. 
         [0008]    In some embodiments of the present invention, improved methods, apparatus and kits are provided for detecting and diagnosing a biological condition in a patient. 
         [0009]    In other embodiments of the present invention, a method and kit is described for providing rapid detection of biological moieties in a sample from a patient. 
         [0010]    In further embodiments of the present invention, a method and kit is disclosed for providing detection of biological moieties in a small fluid sample from a patient. 
         [0011]    There is thus provided according to an embodiment of the present invention, a kit for evaluating a biological condition in a patient, the kit 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. Additionally, according to an embodiment of the present invention, the kit further comprises;   d) instructions for using the kit.       
 
         [0016]    Furthermore, according to an embodiment of the present invention, the disposable element is a disposable cartridge. 
         [0017]    Moreover, according to an embodiment of the present invention, the disposable cartridge is a disposable microfluidics cartridge. 
         [0018]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least one of the following elements:
       a) a reservoir;   b) a pump;   c) a valve;   d) a conduit;   e) a motor;   f) a miniaturized flow cell;   g) a transport channel;   h) a microfluidic element;   i) a compressed gas holding element;   j) a compressed gas releasing element;   k) a nozzle element;   l) a mixing element;   m) a bellows element;   n) software adapted to activate said elements according to a specific sequence; and   o) hardware to activate said elements according to a specific sequence.       
 
         [0034]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least two of the elements. 
         [0035]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least three of the elements. 
         [0036]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least four of the elements. 
         [0037]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least five of the elements. 
         [0038]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least ten of the elements. 
         [0039]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least twenty of the elements. 
         [0040]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least thirty of the elements. 
         [0041]    According to an embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with one hour. 
         [0042]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with thirty minutes. 
         [0043]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with fifteen minutes. 
         [0044]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with ten minutes. 
         [0045]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with five minutes. 
         [0046]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with one minute. 
         [0047]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with thirty seconds. 
         [0048]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with ten seconds. 
         [0049]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with one second. 
         [0050]    There is thus provided according to an embodiment of the present invention, a microfluidics assay kit for performing a rapid biological assay, the kit 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.       
 
         [0053]    There is thus provided according to an embodiment of the present invention, a microfluidics assay kit for performing a rapid assay of a biological entity, the kit 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. 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.   
               
               
 
         [0067]    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 (CD163); 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.   
               
 
         [0079]    Additionally, 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.       
 
         [0082]    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. 
         [0083]    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. 
         [0084]    According to another embodiment of the present invention, the target moiety includes a CD64 surface antigen on neutrophils. 
         [0085]    Additionally, according to a further embodiment of the present invention, the positive control moiety includes monocytes and the negative control includes lymphocytes. 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. 
         [0086]    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. 
         [0087]    Yet further, according to an embodiment of the present invention, the at least one reference composition includes beads. 
         [0088]    Additionally, according to an embodiment of the present invention, the beads include polystyrene microbeads. 
         [0089]    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. 
         [0090]    Furthermore, according to an embodiment of the present invention, the first fluorescent signal includes FITC and the second fluorescent signal includes Starfire Red fluor. 
         [0091]    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.       
 
         [0096]    Furthermore, according to an embodiment of the present invention, the biomarker is a sepsis biomarker. 
         [0097]    Moreover, according to an embodiment of the present invention, the biomarker is CD64 or CD163. 
         [0098]    Additionally, according to an embodiment of the present invention, the sample is a blood sample. 
         [0099]    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. 
         [0100]    Further, according to an embodiment of the present invention, the binding moiety is an antibody. 
         [0101]    According to an embodiment of the present invention, the software is capable of recognizing a specific lot of fluorescently-labeled particles. 
         [0102]    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. 
         [0103]    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. 
         [0104]    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. 
         [0105]    Furthermore, according to an embodiment of the present invention, the binding moiety is anti-CD64 antibody and the first cell population includes polymorphonuclear leukocytes. 
         [0106]    Yet further, according to an embodiment of the present invention, the second cell population includes monocytes. 
         [0107]    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. 
         [0108]    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.   
               
 
         [0120]    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.       
 
         [0123]    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.       
 
         [0126]    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.       
 
         [0131]    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. 
         [0132]    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. 
         [0133]    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.       
 
         [0136]    Additionally, according to an embodiment of the present invention, the sepsis marker is CD64. 
         [0137]    Furthermore, according to an embodiment of the present invention, a second sepsis marker is CD163. 
         [0138]    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. 
         [0139]    Further, according to an embodiment of the present invention, the sepsis marker is CD64 and the second sepsis marker is CD163. 
         [0140]    Additionally, according to an embodiment of the present invention, the binding moiety is an antibody. 
         [0141]    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. 
         [0142]    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. 
         [0143]    According to another embodiment of the present invention, the particles include the same fluorescent label as the fluorescently-labeled binding moiety. 
         [0144]    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. 
         [0145]    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. 
         [0146]    Notably, according to an embodiment of the present invention, the calibration is completed in less than 5 minutes. 
         [0147]    According to some embodiments, the particles are microbeads. 
         [0148]    Additionally, according to an embodiment of the present invention, the method is performed in less than 15 minutes. 
         [0149]    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. 
         [0150]    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 
         [0151]    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. 
           [0152]    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. 
           [0153]    In the drawings: 
           [0154]      FIG. 1  is a simplified schematic illustration showing an apparatus for detecting a biological condition, in accordance with an embodiment of the present invention; 
           [0155]      FIG. 2  is a simplified flow chart of a method for detecting a biological condition, in accordance with an embodiment of the present invention; 
           [0156]      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; 
           [0157]      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; 
           [0158]      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; 
           [0159]      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; 
           [0160]      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; 
           [0161]      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; 
           [0162]      FIG. 6  is a simplified flow chart of a method for differentiating between different particles, in accordance with an embodiment of the present invention; 
           [0163]      FIG. 7  is a graphical output of fluorescence from reference beads in eight wavebands, in accordance with an embodiment of the present invention; 
           [0164]      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; 
           [0165]      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; 
           [0166]      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; and 
           [0167]      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. 
       
    
    
       [0168]    In all the figures similar reference numerals identify similar parts. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0169]    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. 
         [0170]    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. 
         [0171]    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. D669191 S1, US20120266986 A1, EP1846159 A2, US2012275972, W011094577A, US2007292941A and EP1263533 B1. 
         [0172]    Reference is now made to  FIG. 1 , which is a simplified schematic illustration showing an apparatus  100  for detecting a biological condition, in accordance with an embodiment of the present invention. 
         [0173]    Apparatus  100  is a kit comprising a cartridge  102  and a number of chemical/biochemical reactants termed herein, treatment compositions. 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. 
         [0174]    Apparatus  100  comprises a specimen receiving element  118 , adapted to transfer the specimen to a sample composition chamber  104 . The sample composition chamber comprises on 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 . 
         [0175]    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 kit 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. 
         [0176]    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 (e.g., 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.       
 
         [0183]    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. 
         [0184]    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. 
         [0185]    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. 
         [0186]    Cartridge  102  further comprises at least one transfer element  113  in fluid communication with treatment chamber  112  and with an evaluation chamber  114 . 
         [0187]    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. 
         [0188]    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. 
               
             
          
           
               
                   
                   
                   
                 Typical Prior 
                   
                   
               
               
                   
                   
                   
                 Art 
                 This 
               
               
                   
                   
                 Relevant 
                 Laboratory 
                 invention 
               
               
                   
                   
                 Figures in 
                 Turnaround 
                 Turnaround 
               
               
                   
                 Type of 
                 this 
                 time (TAT)- 
                 time 
               
               
                 Application 
                 Test 
                 invention 
                 see references 
                 (TAT) 
                 References 
               
               
                   
               
               
                 Application #1 - 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 U.S. Pat. No. 8,116,984, 
               
               
                 CD64 Infection &amp; 
                 Marker 
                 3-5D 
                   
                   
                 Davis, BH et al., 
               
               
                 Sepsis 
                   
                   
                   
                   
                 (2006) 
               
               
                 1 - Fetal Hemoglobin 
                 Plasma 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Dziegiel et al. 
               
               
                 Test 
                 Protein 
                 6-8D 
                   
                   
                 (2006) 
               
               
                 2 - Low Platelet 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Segal, H. C., et al. 
               
               
                 Count 
                 Marker 
                 3-5D 
                   
                   
                 (2005): 
               
               
                 3 - Resolving BLAST 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Guerti, K., et al. 
               
               
                 Flag for hematology 
                 Marker 
                 3-5D 
                   
                   
               
               
                 Lab 
               
               
                 4 - CD34 Stem Cell 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Sutherland et al. 
               
               
                 Enumeration Assay 
                 Marker 
                 3-5D 
                   
                   
                 (1996) 
               
               
                 5 - Platelets 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Graff et al. (2002) 
               
               
                 Activation Assay 
                 Marker 
                 3-5D 
                   
                   
                 Divers, S. G., et 
               
               
                 CD62 
                   
                   
                   
                   
                 al. (2003) 
               
               
                 6 - D-dimer (Bead 
                 Plasma 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Stein et al. (2004) 
               
               
                 based protein) 
                 Protein 
                 6-8D 
                   
                   
                 Rylatt, D. B., et 
               
               
                   
                   
                   
                   
                   
                 al. (1983): 
               
               
                 7 - Chorioamnioitis 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Hillier et al. 
               
               
                 CD64 
                 Marker 
                 3-5D 
                   
                   
                 (1988) 
               
               
                 8 - CD20 Cell 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Rawstron et al. 
               
               
                 Quantitation 
                 Marker 
                 3-5D 
                   
                   
                 (2001) 
               
               
                 (Therapy Monitoring 
                   
                   
                   
                   
                 Cheson et al. 
               
               
                   
                   
                   
                   
                   
                 (1996) 
               
               
                 9 - CD52 Cell 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Rawstron et al. 
               
               
                 quantitation (Therapy 
                 Marker 
                 3-5D 
                   
                   
                 (2001) 
               
               
                 Monitoring) 
               
               
                 10 - Circulating 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Cristofanilli et al. 
               
               
                 Tumor Cells 
                 Marker 
                 3-5D 
                   
                   
                 (2004 
               
               
                 11 - Reticulated 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Matic et al. 
               
               
                 Platelet Assay 
                 Marker 
                 3-5D 
                   
                   
                 (1998) 
               
               
                   
                   
                   
                   
                   
                 Ault et al (1993) 
               
               
                   
                   
                   
                   
                   
                 Wang et al. 
               
               
                   
                   
                   
                   
                   
                 (2002) 
               
               
                 12 - Bacteria 
                   
                   
                 4 hours 
                 10 minutes 
                 Blajchman et al 
               
               
                 Detection in platelet 
                   
                   
                   
                   
                 (2005) 
               
               
                 packs 
                   
                   
                   
                   
                 McDonald et al. 
               
               
                   
                   
                   
                   
                   
                 (2005) 
               
               
                 13 - Platelet 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Michelson (1996) 
               
               
                 Associated 
                 Marker 
                 3-5D 
                   
                   
               
               
                 Antibodies 
               
               
                 14 - Residual 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Bodensteiner, 
               
               
                 Leukocyte Count in 
                 Marker 
                 3-5D 
                   
                   
                 (2003) 
               
               
                 blood products 
               
               
                 15 - CD4 HIV AIDS 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Rodriguez (2005). 
               
               
                   
                 Marker 
                 3-5D 
                   
                   
                 Dieye et al. 
               
               
                   
                   
                   
                   
                   
                 (2005) 
               
               
                 16 - Leukemia Panels -  
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Drexler et al 
               
               
                 Very complex 
                 Marker 
                 3-5D 
                   
                   
                 (1986) 
               
               
                 17 - Bladder Cancer 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Ramakumar et al 
               
               
                 Screening in Urine -  
                 Marker 
                 3-5D 
                   
                   
                 (1999) 
               
               
                 Urine sample 
                   
                   
                   
                   
                 Lotan et al. 
               
               
                   
                   
                   
                   
                   
                 (2009) 
               
               
                 18 - HLA DR Sepsis 
                 Surface 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Hershman et al. 
               
               
                 and 
                 Marker 
                 3-5D 
                   
                   
                 (2005) 
               
               
                 Immunosuppression 
                   
                   
                   
                   
                 Perry et al (2003) 
               
               
                 19 - RECAF Protein 
                 Plasma 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Moro et al. 
               
               
                 for Canine and other 
                 Protein 
                 6-8D 
                   
                   
                 (2005). 
               
               
                 Cancers 
               
               
                 20 - CytoImmun -  
                   
                   
                 4 hours 
                 10 minutes 
                 Hilfrich et al. 
               
               
                 Cervical Screening 
                   
                   
                   
                   
                 (2008) 
               
               
                 21 - Procalcitonin 
                 Plasma 
                 FIGS. 1-2 and 
                 4 hours 
                 10 minutes 
                 Assicot et al. 
               
               
                 (Bead Based Protein) + 
                 Protein 
                 6-8D 
                   
                   
                 (1993) 
               
               
                 Feasibility 
                   
                   
                   
                   
                 Christ-Crain et al. 
               
               
                   
                   
                   
                   
                   
                 (2004) 
               
               
                   
               
             
          
         
       
     
         [0189]    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. 
         [0190]    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. 
         [0191]    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. 
         [0192]    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. 
         [0193]    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 kit. Additionally or alternatively, stirbars may be disposed in any of the other chambers in the apparatus. 
         [0194]    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. 
         [0195]    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. 
         [0196]    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. 
         [0197]    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. 
         [0198]    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. 
         [0199]    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. 
         [0200]    Thereafter, in a second sample transfer step  210 , the sample is transferred from the sample composition chamber into the treatment chamber. 
         [0201]    According to some embodiments, step  210  may be performed before steps  204 - 208 . If required, an optional mixing step  212  to the contents of the treatment chamber may be performed. 
         [0202]    In a transferring step  214 , the contents of the treatment chamber are transferred to the evaluation chamber. 
         [0203]    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.       
 
         [0214]    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. 
         [0215]    The results of the evaluation step are then outputted in a results outputting step  218 . 
         [0216]    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 . 
         [0217]    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. 
         [0218]    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. 
         [0219]    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. 
         [0220]    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. 
         [0221]    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 . 
         [0222]    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. 
         [0223]    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. 
         [0224]    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. 
         [0225]    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. 
         [0226]    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. 
         [0227]    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. 
         [0228]    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. 
         [0229]    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. 
         [0230]    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. 
         [0231]    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 . 
         [0232]    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. 
         [0233]    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 . 
         [0234]    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). 
         [0235]    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. 
         [0236]    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 . 
         [0237]      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. 
         [0238]    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 . 
         [0239]      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 . 
         [0240]    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. 
         [0241]    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). 
         [0242]    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. 
         [0243]    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 crosscorrelation 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 crosscorrelations  606 . One or more of these derived time series may be used to determine event locations. 
         [0244]    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. 
         [0245]    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 . 
         [0246]    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. 
         [0247]      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. 
         [0248]    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. 
         [0249]      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. 
         [0250]      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. 
         [0251]      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. 
       Example 
     Application No. 1-CD64 Infection &amp; Sepsis 
       [0252]    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). 
         [0253]    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 . 
         [0254]    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 . 
         [0255]    These two steps combined with mixing step  206  take around four minutes using cartridge  102  of the present invention. 
         [0256]    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. 
         [0257]    The evaluation chamber  114  is configured and constructed for one or more evaluation steps  216 . 
         [0258]    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. 
         [0259]    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. 
         [0260]    From a user point of view, the following steps are performed: 
         [0261]    1) The user adds drop of blood to the cartridge  102  and seals it. (10 μL are metered out by micro fluidics).
       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 
               
             
          
           
               
                 Step 
                 Description 
                 Volume (uL) 
                 Duration (min) 
                 comments 
               
               
                   
               
               
                 1 
                 Mixing blood and 
                 Blood- 10 
                 4 
                   
               
               
                   
                 antibodies 
                 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  
               
               
                   
               
             
          
         
       
     
         [0269]    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. 
         [0270]    According to some embodiments, the readout may comprise an optoelectronics core, which enables identification and detection of fluorescent signals. 
         [0271]    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. 
         [0272]    The contents of these publications are incorporated by reference herein where appropriate for teachings of additional or alternative details, features and/or technical background. 
         [0273]    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. 
       REFERENCES 
       [0274]    Assicot, Marcel, et al. “High serum procalcitonin concentrations in patients with sepsis and infection.”  The Lancet  341.8844 (1993): 515-518. 
         [0275]    Aulesa, C., et al. “Validation of the Coulter LH 750 in a hospital reference laboratory.”  Laboratory Hematology  9.1 (2003): 15-28. 
         [0276]    Ault, Kenneth A. “Flow cytometric measurement of platelet function and reticulated platelets.”  Annals of the New York Academy of Sciences  677.1 (1993): 293-308. 
         [0277]    Blajchman, Morris A., et al. “Bacterial detection of platelets: current problems and possible resolutions.”  Transfusion medicine reviews  19.4 (2005): 259-272. 
         [0278]    Bodensteiner, David C. “A flow cytometric technique to accurately measure post-filtration white blood cell counts.”  Transfusion  29.7 (1989): 651-653. 
         [0279]    Cheson, Bruce D., et al. “National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment.”  Blood  87.12 (1996): 4990-4997. 
         [0280]    Christ-Crain, Mirjam, et al. “Effect of procalcitonin-guided treatment on antibiotic use and outcome in lower respiratory tract infections: cluster-randomised, single-blinded intervention trial.”  Lancet  363.9409 (2004): 600-607. 
         [0281]    Cristofanilli, Massimo, et al. “Circulating tumor cells, disease progression, and survival in metastatic breast cancer.”  New England Journal of Medicine  351.8 (2004): 781-791. 
         [0282]    Davis, Bruce H., et al. “Neutrophil CD64 is an improved indicator of infection or sepsis in emergency department patients.” Archives of pathology &amp; laboratory medicine 130.5 (2006): 654-661. 
         [0283]    Dieye, Tandakha Ndiaye, et al. “Absolute CD4 T-cell counting in resource-poor settings: direct volumetric measurements versus bead-based clinical flow cytometry instruments.”  JAIDS Journal of Acquired Immune Deficiency Syndromes  39.1 (2005): 32-37. 
         [0284]    Divers, S. G., et al. “Quantitation of CD62, soluble CD62, and lysosome-associated membrane proteins 1 and 2 for evaluation of the quality of stored platelet concentrates.”  Transfusion  35.4 (2003): 292-297. 
         [0285]    Drexler, Hans G., et al. “Diagnostic value of immunological leukemia phenotyping.”  Acta haematologica  76.1 (1986): 1-8. 
         [0286]    Dziegiel, Morten Hanefeld, Leif Kofoed Nielsen, and Adela Berkowicz. “Detecting fetomaternal hemorrhage by flow cytometry.”  Current opinion in hematology  13.6 (2006): 490. 
         [0287]    Fischer, Johannes C., et al. “Reducing costs in flow cytometric counting of residual white blood cells in blood products: utilization of a single platform bead free flow rate calibration method.”  Transfusion  51.7 (2011): 1431-1438. 
         [0288]    Graff, Jochen, et al. “Close relationship between the platelet activation marker CD62 and the granular release of platelet-derived growth factor.”  Journal of Pharmacology and Experimental Therapeutics  300.3 (2002): 952-957. 
         [0289]    Guerti, K., et al. “Performance evaluation of the PENTRA 60C+ automated hematology analyzer and comparison with the ADVIA 2120 .” International journal of laboratory hematology  31.2 (2009): 132-141. 
         [0290]    Hawkins, Robert C. “Laboratory turnaround time.”  The Clinical Biochemist Reviews  28.4 (2007): 179. 
         [0291]    Hershman, M. J., et al. “Monocyte HLA-DR antigen expression characterizes clinical outcome in the trauma patient.”  British Journal of Surgery  77.2 (2005): 204-207. 
         [0292]    Hilfrich, Ralf, and Jalil Hariri. “Prognostic relevance of human papillomavirus L1 capsid protein detection within mild and moderate dysplastic lesions of the cervix uteri in combination with p16 biomarker.”  Analytical and Quantitative Cytology and Histology  30.2 (2008): 78-82. 
         [0293]    Hillier, Sharon L., et al. “A case—control study of chorioamnionic infection and histologic chorioamnionitis in prematurity.” New England Journal of Medicine 319.15 (1988): 972-978. 
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