PATENT DOCUMENT

Abstract:
The present invention provides self-contained systems, apparatus and methods for determining a chemical state, the system includes a stationary cartridge for performing the assay therein, the cartridge adapted to house at least one reagent adapted to react with a sample; and at least one reporter functionality adapted to report a reaction of the at least one reagent with the sample to report a result of the assay, a mechanical controller including a first urging means adapted to apply a force externally onto the cartridge to release the at least one reagent; and at least one second urging means adapted to apply a removable force to induce fluidic movement in a first direction in the cartridge and upon removal of the force causing fluidic movement in an opposite direction to the first direction, an optical reader adapted to detect the reaction and a processor adapted to receive data from the optical reader and to process the data to determine said chemical state.

Full Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present invention claims priority from U.S. provisional patent application 61/737,854, to Kasdan et al, filed on Dec. 17, 2012, U.S. provisional patent application 61/737,856, to Kasdan et al., filed on Dec. 17, 2012 and from U.S. patent application Ser. No. 13/716,246 filed on Dec. 17, 2012, incorporated herein by reference. 
     
    
     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 kits are provided for detecting and diagnosing a biological condition in a patient. 
         [0008]    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. 
         [0009]    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. 
         [0010]    There is thus provided according to an embodiment of the present invention, a self-contained system for performing an assay for determining a chemical state, the system including;
       a) a stationary cartridge for performing the assay therein, the cartridge adapted to house at least one reagent adapted to react with a sample; and at least one reporter functionality adapted to report a reaction of the at least one reagent with the sample to report a result of the assay;   b) a mechanical controller including;
           i. a first urging means adapted to apply a force externally onto the cartridge to release the at least one reagent;   ii. at least one second urging means adapted to apply a removable force to induce fluidic movement in a first direction in the cartridge and upon removal of the force causing fluidic movement in an opposite direction to the first direction;   
           c) an optical reader adapted to detect the reaction; and   d) a processor adapted to receive data from the optical reader and to process the data to determine the chemical state.       
 
         [0017]    Additionally, according to an embodiment of the present invention, the cartridge further includes an alignment means adapted to align a reading channel on the cartridge for a detection of the least one reporter functionality. 
         [0018]    Furthermore, according to an embodiment of the present invention, the cartridge further comprises a plurality of fluidic open channels, all the channels in liquid communication with each other. 
         [0019]    Moreover, according to an embodiment of the present invention, the cartridge is adapted to be sealed after receiving a fluid specimen and to pass a predetermined quantity of the sample through at least part of the plurality of fluidic open channels. 
         [0020]    Further, according to an embodiment of the present invention, the cartridge further includes at least one inflatable deformable elastic chamber adapted to apply at least one of a negative pressure and a positive pressure in the fluidic channels. 
         [0021]    Additionally, according to an embodiment of the present invention, the at least one deformable elastic chamber is adapted to further contact the at least one reagent stored in a sealed on-board storage chamber with a predetermined quantity of the sample in a reaction chamber to induce the reaction. 
         [0022]    Further, according to an embodiment of the present invention, the a first urging means is disposed proximal to the on-board storage chamber such that upon movement is adapted to break a frangible seal on the storage chamber. 
         [0023]    Yet further, according to an embodiment of the present invention, the alignment means adapted to align with a reading channel on the cartridge for a detection of a reaction in the predetermined quantity of the sample. 
         [0024]    Additionally, according to an embodiment of the present invention, some of the plurality of fluidic open channels is of a cross-section of 0.1 to 2 mm 2 . 
         [0025]    Notably, according to an embodiment of the present invention, the predetermined quantity is of a volume of 10 to 500 microliters. 
         [0026]    Furthermore, according to an embodiment of the present invention, the cartridge is adapted to contact a plurality of on-board reagents with the at least one of the sample and a reaction product. 
         [0027]    In some cases, according to an embodiment of the present invention, the cartridge is adapted to induce cascaded sequential reactions of the plurality of on-board reagents, with the at least one of the sample and the reaction product. 
         [0028]    Additionally, according to an embodiment of the present invention, the cartridge includes at least one reaction chamber of a volume of 200 to 10000 microliters. 
         [0029]    Furthermore, 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 a temperature of the reaction. 
         [0030]    Additionally, according to an embodiment of the present invention, the cartridge has a shelf-life of 6 to 24 months. 
         [0031]    Importantly, according to an embodiment of the present invention, the cartridge is valveless. 
         [0032]    Notably, according to an embodiment of the present invention, the assay is a flow cytometric assay. 
         [0033]    Additionally, according to an embodiment of the present invention, the chemical state is a biochemical state. 
         [0034]    Furthermore, according to an embodiment of the present invention, the biochemical state is indicative of a biological condition. 
         [0035]    Additionally, according to an embodiment of the present invention, the sample is a biological sample. 
         [0036]    In some cases, according to an embodiment of the present invention, the biological sample is a bodily sample. 
         [0037]    Moreover, according to an embodiment of the present invention, the bodily sample is selected from a the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid (CSF), serous fluid, peritoneal fluid and synovial fluid blood, urine, plasma, serum and saliva. 
         [0038]    Additionally, according to an embodiment of the present invention, the cartridge is valveless. 
         [0039]    Further, according to an embodiment of the present invention, the cartridge is a disposable microfluidics cartridge. 
         [0040]    Yet further, according to an embodiment of the present invention, the sample is introduced to the cartridge via capillary action. 
         [0041]    Additionally, 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.       
 
         [0056]    Additionally, according to an embodiment of the present invention, the at least one reagent 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.       
 
         [0060]    Moreover, according to an embodiment of the present invention, the at least one reagent disposed in the cartridge includes at least one reference composition including at least one of;
       a. a target signal reference composition; and   b. a reference identifier composition.       
 
         [0063]    Additionally, according to an embodiment of the present invention, the at least one reagent disposed in the cartridge includes at least one of;
       a. a positive control moiety; and   b. a negative control moiety.       
 
         [0066]    Furthermore, according to an embodiment of the present invention, the at least one reagent disposed in the cartridge includes at least one sepsis biomarker. 
         [0067]    There is thus provided according to another embodiment of the present invention, a method for determining a biological condition in a subject, the method including;
       a. incubating a sample from the subject in the system described herein for a predetermined period of time; and   b. receiving an indication responsive to the at least one reporter functionality thereby providing an indication of the biological condition in the subject in accordance with the chemical state.       
 
         [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]    Furthermore, according to an embodiment of the present invention, the indication is quantitative. 
         [0072]    Moreover, according to an embodiment of the present invention, the method is completed within twenty minutes. 
         [0073]    There is thus provided according to another 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 described herein, 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.       
 
         [0076]    There is thus provided according to another 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 in the system as described herein, the 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.       
 
         [0079]    Furthermore, according to an embodiment of the present invention, wherein the sepsis marker is CD64. 
         [0080]    Additionally, according to an embodiment of the present invention, the sepsis marker is CD163. 
         [0081]    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. 
         [0082]    Further, according to an embodiment of the present invention, the sepsis marker is CD64 and the second sepsis marker is CD163. 
         [0083]    There is thus provided according to another embodiment of the present invention, a method for performing an assay for determining a chemical state in a self-contained stationary cartridge, the method including;
       a. introducing a sample into the system described herein;   b. reacting at least one reagent with the sample; and   c. detecting a signal associated with at least one reporter functionality, the at least one reporter functionality adapted to report a reaction of the at least one reagent with the sample, thereby determining the chemical state.       
 
         [0087]    Furthermore, according to an embodiment of the present invention, the method further includes forming at least one product and detecting a signal associated with the product. 
         [0088]    Additionally, according to an embodiment of the present invention the assay is a flow cytometric assay. 
         [0089]    Further, according to an embodiment of the present invention, the chemical state is a biochemical state. 
         [0090]    Further, according to an embodiment of the present invention, the biochemical state is indicative of a biological condition. 
         [0091]    Furthermore, according to an embodiment of the present invention, the sample is a biological sample. 
         [0092]    Additionally, according to an embodiment of the present invention, the biological sample is a bodily sample. 
         [0093]    Furthermore, according to an embodiment of the present invention, the bodily sample is selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid (CSF), serous fluid, peritoneal fluid and synovial fluid. 
         [0094]    Moreover, according to an embodiment of the present invention, the at least one reagent includes at least one of;
       a. a cell surface marker;   b. a cell stain;   c. a reagent bound to a solid support;   d. a chemical indicator; and   e. a biological cell indicator.       
 
         [0100]    Furthermore, according to an embodiment of the present invention, the cell surface marker is selected from the group consisting of CD64, CD4, CD8, a stem cell indicator, a Minimal Residual Disease indicator and a lymphocyte subtype indicator. 
         [0101]    Additionally, according to an embodiment of the present invention, the cell stain is selected from the group consisting of a white blood cell differential indicator, an apoptosis indicator. 
         [0102]    Further, according to an embodiment of the present invention, the reagent bound to the solid support is selected from the group consisting of an immobilized enzyme, an immobilized substrate, a plasma protein bead, an antibody bead, an antigen bead and an ELISA assay. 
         [0103]    Additionally, according to an embodiment of the present invention, the chemical indicator is selected from the group consisting of a color indicator, a turbidity indicator, a pH indicator, an adsorption indicator, an emission indicator and a chemical reaction indicator. 
         [0104]    Furthermore, according to an embodiment of the present invention, the biological cell indicator is selected from the group consisting of a cell cycle stage indicator, a cell proliferation indicator, a cytokine indicator, a metabolic indicator and an apoptosis indicator. 
         [0105]    Further, according to an embodiment of the present invention, the at least one reagent includes at least two reagents. 
         [0106]    Moreover, according to an embodiment of the present invention, the at least two reagents include at least one of;
       a. a cell surface marker and a cell element stain;   b. a cell surface marker and a plasma protein bead assay;   c. a cell surface marker and a solution change marker;   d. a cell element stain and a plasma protein bead assay; and   e. a cell element stain and a solution change marker.       
 
         [0112]    Furthermore, 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. 
         [0113]    There is thus provided according to another embodiment of the present invention, a method for forming a chemical reaction in a stationary cartridge, the method including;
       a. storing at least one composition in the cartridge described herein; and   b. activating at least one inflatable chamber in the cartridge to provide at least one pressure force to the at least one reagent thereby inducing the chemical reaction.       
 
         [0116]    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.       
 
         [0120]    Additionally, according to an embodiment of the present invention, the kit further comprises;
       d) instructions for using the kit.       
 
         [0122]    Furthermore, according to an embodiment of the present invention, the disposable element is a disposable cartridge. 
         [0123]    Moreover, according to an embodiment of the present invention, the disposable cartridge is a disposable microfluidics cartridge. 
         [0124]    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 conduit;   d) a miniaturized flow cell;   e) a transport channel;   f) a microfluidic element;   g) a compressed gas holding element;   h) a compressed gas releasing element;   i) a nozzle element;   j) a mixing element;   k) a bellows element;   i) software adapted to activate said elements according to a specific sequence; and   m) hardware to activate said elements according to a specific sequence.       
 
         [0138]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least two of the elements. 
         [0139]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least three of the elements. 
         [0140]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least four of the elements. 
         [0141]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least five of the elements. 
         [0142]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least ten of the elements. 
         [0143]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least twenty of the elements. 
         [0144]    Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least thirty of the elements. 
         [0145]    According to an embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with one hour. 
         [0146]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with thirty minutes. 
         [0147]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with fifteen minutes. 
         [0148]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with ten minutes. 
         [0149]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with five minutes. 
         [0150]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with one minute. 
         [0151]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with thirty seconds. 
         [0152]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with ten seconds. 
         [0153]    According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with one second. 
         [0154]    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.       
 
         [0157]    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.       
 
         [0160]    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.   
               
 
         [0172]    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;
           iii. a bodily specimen comprising a target moiety;   iv. a positive control moiety; and   v. a negative control moiety;   
           b. an antibody composition comprising at least one of;
           vi. at least one target antibody (CD64 antibody);   vii. at least one positive control identifying antibody (CD163); and   viii. at least one negative control identifying antibody or characteristic; and   
           c. at least one reference composition comprising at least one of;
           ix. a target signal reference composition; and   x. a reference identifier composition.   
               
 
         [0184]    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.       
 
         [0187]    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. 
         [0188]    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. 
         [0189]    According to another embodiment of the present invention, the target moiety includes a CD64 surface antigen on neutrophils. 
         [0190]    Additionally, according to a further embodiment of the present invention, the positive control moiety includes monocytes and the negative control includes lymphocytes. 
         [0191]    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. 
         [0192]    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. 
         [0193]    Yet further, according to an embodiment of the present invention, the at least one reference composition includes beads. 
         [0194]    Additionally, according to an embodiment of the present invention, the beads include polystyrene microbeads. 
         [0195]    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. 
         [0196]    Furthermore, according to an embodiment of the present invention, the first fluorescent signal includes FITC and the second fluorescent signal includes Starfire Red fluor. 
         [0197]    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.       
 
         [0202]    Furthermore, according to an embodiment of the present invention, the biomarker is a sepsis biomarker. 
         [0203]    Moreover, according to an embodiment of the present invention, the biomarker is CD64 or CD163. 
         [0204]    Additionally, according to an embodiment of the present invention, the sample is a blood sample. 
         [0205]    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. 
         [0206]    Further, according to an embodiment of the present invention, the binding moiety is an antibody. 
         [0207]    According to an embodiment of the present invention, the software is capable of recognizing a specific lot of fluorescently-labeled particles. 
         [0208]    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. 
         [0209]    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. 
         [0210]    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. 
         [0211]    Furthermore, according to an embodiment of the present invention, the binding moiety is anti-CD64 antibody and the first cell population includes polymorphonuclear leukocytes. 
         [0212]    Yet further, according to an embodiment of the present invention, the second cell population includes monocytes. 
         [0213]    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. 
         [0214]    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;
           iv. at least one target antibody;   v. at least one positive control identifying antibody; and   vi. at least one negative control identifying antibody or characteristic; and   
           c. at least one reference composition comprising at least one of;
           vii. a target antibody reference composition; and   viii. a reference identifier composition.   
               
 
         [0226]    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.       
 
         [0229]    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.       
 
         [0232]    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.       
 
         [0237]    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. 
         [0238]    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. 
         [0239]    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.       
 
         [0242]    Additionally, according to an embodiment of the present invention, the sepsis marker is CD64. 
         [0243]    Furthermore, according to an embodiment of the present invention, a second sepsis marker is CD163. 
         [0244]    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. 
         [0245]    Further, according to an embodiment of the present invention, the sepsis marker is CD64 and the second sepsis marker is CD163. 
         [0246]    Additionally, according to an embodiment of the present invention, the binding moiety is an antibody. 
         [0247]    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. 
         [0248]    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. 
         [0249]    According to another embodiment of the present invention, the particles include the same fluorescent label as the fluorescently-labeled binding moiety. 
         [0250]    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. 
         [0251]    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. 
         [0252]    Notably, according to an embodiment of the present invention, the calibration is completed in less than 5 minutes. 
         [0253]    According to some embodiments, the particles are microbeads. 
         [0254]    Additionally, according to an embodiment of the present invention, the method is performed in less than 15 minutes. 
         [0255]    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. 
         [0256]    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 
         [0257]    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. 
           [0258]    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. 
           [0259]    In the drawings: 
           [0260]      FIG. 1A  is a simplified three dimensional front view of a system for detecting a biological condition, in accordance with an embodiment of the present invention; 
           [0261]      FIG. 1B  is a simplified three dimensional inner front view of a reader assembly for detecting a biological condition, in accordance with an embodiment of the present invention; 
           [0262]      FIG. 1C  is a simplified three dimensional inner rear view of a reader assembly for detecting a biological condition, in accordance with an embodiment of the present invention; 
           [0263]      FIG. 2A  is a simplified blown up diagram of an optical reader assembly for detecting a biological condition, in accordance with an embodiment of the present invention; 
           [0264]      FIG. 2B  is another simplified blown up diagram of a photomultiplier tube of the optical reader assembly for detecting a biological condition, in accordance with an embodiment of the present invention; 
           [0265]      FIG. 3A  shows a reader optics assembly, a cartridge handling unit, and a forward scatter detection unit, in accordance with an embodiment of the present invention; 
           [0266]      FIG. 3B  shows a right side view of a reader optics assembly, in accordance with an embodiment of the present invention; 
           [0267]      FIG. 3C  shows a left side view of a reader optics assembly, in accordance with an embodiment of the present invention; 
           [0268]      FIG. 3D  is a forward scatter detection assembly, in accordance with an embodiment of the present invention; 
           [0269]      FIG. 3E  is a side view of the forward scatter detection assembly, in accordance with an embodiment of the present invention; 
           [0270]      FIG. 4A  shows a cutaway view of a reader assembly, in accordance with an embodiment of the present invention; 
           [0271]      FIG. 4B  shows an exploded right side view of a reader assembly, in accordance with an embodiment of the present invention; 
           [0272]      FIG. 4C  shows a left side blown up view of the reader assembly, in accordance with an embodiment of the present invention; 
           [0273]      FIG. 4D  shows a rear view of a cartridge handling unit (CHU), in accordance with an embodiment of the present invention; 
           [0274]      FIG. 4E  shows a front view of a cartridge handling unit (CHU), in accordance with an embodiment of the present invention; 
           [0275]      FIG. 5  shows an exploded view of a reader optics assembly, in accordance with an embodiment of the present invention; 
           [0276]      FIG. 6  is a simplified illustration of a disposable cartridge of the system of  FIG. 1A , in accordance with an embodiment of the present invention; 
           [0277]      FIG. 7A  is a simplified schematic illustration of an optical arrangement of a reader optics assembly, in accordance with an embodiment of the present invention; 
           [0278]      FIG. 7B  is another simplified schematic illustration of optical arrangement of a reader optics assembly, in accordance with an embodiment of the present invention; 
           [0279]      FIG. 8A  is a schematic representation of one example of multi-wavelength excitation in the optical unit of  FIG. 7A  or  7 B, in accordance with an embodiment of the present invention; 
           [0280]      FIG. 8B  shows a graphical output of transmission as a function of wavelength for a dichroic filter of  FIG. 7B , employing the multi-wavelength excitation of  FIG. 8A , in accordance with an embodiment of the present invention; 
           [0281]      FIG. 8C  is a schematic representation of part of the optical unit employing multi-wavelength excitation of  FIG. 8A  and the dichroic filter of  FIG. 5B , in accordance with an embodiment of the present invention. 
           [0282]      FIG. 9A  is a schematic view of a sampling cartridge of the system of  FIG. 1A , in accordance with an embodiment of the present invention; 
           [0283]      FIG. 9B  shows a schematic view of disposable cartridge in flow-cytometer device, in accordance with an embodiment of the present invention; 
           [0284]      FIG. 10  is a simplified flowchart of a method for rapid determination of a medical condition, in accordance with an embodiment of the present invention; 
           [0285]      FIG. 11  is a three-dimensional graph showing the optical output over time of reference beads (RM) relative to a sample from a human patient (PMN), in accordance with an embodiment of the present invention; 
           [0286]      FIGS. 12A-12C  show graphs of optical outputs over time of the reference beads and the sample from a human patient, in accordance with an embodiment of the present invention; 
           [0287]      FIG. 13A  is an outer side view of a cartridge assembly, in accordance with an embodiment of the present invention; 
           [0288]      FIG. 13B  is an inner side view of a cartridge assembly, in accordance with an embodiment of the present invention; 
           [0289]      FIG. 14A-14O  show a sequence of process events in a cartridge assembly, in accordance with an embodiment of the present invention; 
           [0290]      FIG. 15  is a schematic illustration of a micro flow spectrometer reading, in accordance with an embodiment of the present invention; 
           [0291]      FIG. 16  is a flow chart of a method for optical processing, in accordance with an embodiment of the present invention; 
           [0292]      FIGS. 17A-17B  are schematic illustrations of steps of use of a graphical user interface, in accordance with an embodiment of the present invention; 
           [0293]      FIG. 18  is a cartridge block diagram showing a role of signal processing software, in accordance with an embodiment of the present invention; 
           [0294]      FIG. 19A  is a flow chart of an algorithm for biological detection, in accordance with an embodiment of the present invention; 
           [0295]      FIG. 19B  is a flow chart of an algorithm for biological detection, in accordance with an embodiment of the present invention; 
           [0296]      FIGS. 20A-20B  shows bandwidth leveled and smoothed arrays, in accordance with an embodiment of the present invention; 
           [0297]      FIGS. 21A-21C  are schematics for solving a fluor decomposition of an observed signal, in accordance with an embodiment of the present invention; 
           [0298]      FIGS. 22A-22B  is a graphical comparison of system performance with FITC beads with MESF detection versus FACS, in accordance with an embodiment of the present invention; 
           [0299]      FIGS. 23A-23B  show graphical displays of linearity of system performance with Alexa 488 MESF, in accordance with an embodiment of the present invention; 
           [0300]      FIG. 24  is a three-dimensional graph showing the optical output over time of a CD4-CD8 assay, in accordance with an embodiment of the present invention; 
           [0301]      FIG. 25  is a graphical display showing a cluster analysis of a CD4-CD8 assay, in accordance with an embodiment of the present invention; 
           [0302]      FIGS. 26-27  are graphical displays showing cluster separations of the cluster analysis of  FIG. 25 , in accordance with an embodiment of the present invention; and 
           [0303]      FIG. 28  is a comparison table of different array options, in accordance with an embodiment of the present invention. 
           [0304]      FIG. 29A  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; 
           [0305]      FIG. 29B  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; 
           [0306]      FIG. 30  is a scatterplot matrix of the four fluors signatures showing four distinct event groups, in accordance with an embodiment of the present invention; 
           [0307]      FIG. 31A  is a histogram of data of Starfire Red (SFR) signature values, in accordance with an embodiment of the present invention; 
           [0308]      FIG. 31B  is a plot of a polynomial and first and second derivative thereof of the histogram shown in  FIG. 31A , in accordance with an embodiment of the present invention; 
           [0309]      FIG. 32A  is a histogram of data of PE488 signature values, in accordance with an embodiment of the present invention; 
           [0310]      FIG. 32B  shows a polynomial fitted to the histogram in  FIG. 32A  as well as corresponding first and second derivatives, in accordance with an embodiment of the present invention; 
           [0311]      FIG. 33A  is a histogram of data of PEAF488 signature values, in accordance with an embodiment of the present invention; 
           [0312]      FIG. 33B  shows a polynomial fitted to the histogram in  FIG. 33A  as well as corresponding first and second derivatives, in accordance with an embodiment of the present invention. 
           [0313]      FIG. 34A  is a histogram of data of Diode 1 channel signature values, in accordance with an embodiment of the present invention; and 
           [0314]      FIG. 34B  shows the polynomial fitted to the histogram in  FIG. 34A  as well as the corresponding first and second derivatives, in accordance with an embodiment of the present invention. 
       
    
    
       [0315]    In all the figures similar reference numerals identify similar parts. 
       DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0316]    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. 
         [0317]    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. 
         [0318]    Reference is now made to  FIG. 1A , which is a simplified three dimensional front view of a system  101  comprising a reader assembly  100  and a cartridge  110  for detecting a biological condition, in accordance with an embodiment of the present invention. 
         [0319]    Shown in  FIG. 1A  are the reader assembly  100  and the cartridge  110 . 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 touchscreen  115 , which is also used to control operation of the reader. 
         [0320]      FIG. 1B  shows a simplified three dimensional inner front view  103  of reader assembly  100  for detecting a biological condition, in accordance with an embodiment of the present invention. 
         [0321]    The internal components of the reader assembly are shown in  FIG. 1B . There is seen left side view  120 , showing an ITX computer,  122 , a Galil motor controller,  124 , an electronics power supply  126 , cartridge,  110 , inserted into a cartridge handling unit (CHU)  128  and a forward scatter detector  130 . Also seen, is a right side view  140  showing reader optics  142 , a data acquisition board  144  and a general electronics printed circuit board  146 . 
         [0322]    Reference is now made to  FIG. 2A , which is a simplified blown up diagram of a reader optics assembly  200  for detecting a biological condition, in accordance with an embodiment of the present invention.  FIG. 2B  is another simplified blown up diagram of a photomultiplier tube  250  of the optical reader assembly for detecting a biological condition, in accordance with an embodiment of the present invention. 
         [0323]      FIG. 2A  shows the main modular components of a reader optics assembly  200 . A complete side view  220  of the optical assembly is seen, in addition to a top view  222 . A laser unit  203  includes a laser and beam expander  223  in its heat sink assembly  221 . The assembly further comprises an excitation and emission collection optics  204 . The reader optics assembly also comprises a photomultiplier assembly  202 , a laser mirror cover  205 , a PMT mirror cover  206 , a modified M6 set screw  207 , a box clamp  208 , and various screws  209 - 211 . The reader optics assembly is assembled as shown in  FIG. 2A . 
         [0324]      FIG. 2B  shows details of the PMT assembly. A side view and an end view of the PMT assembly are shown as side view  270  and end view  272  respectively. The major elements of the PMT assembly include a PMT box  251 , a PMT grating assembly  252 , a PMT bridge assembly  255 , a PMT cover  258 , a PMT unit  259 , a PMT lens assembly  260 , a PMT pinhole nut  261 , a pinhole  262 , a pinhole hood  263  and an adjustment bar  265 . 
         [0325]      FIG. 3A  shows a reader optics assembly  310 , a cartridge handling unit  312  and a forward scatter detection unit  314 , in accordance with an embodiment of the present invention. 
         [0326]      FIG. 3B  shows a right side view of a complete reader optics assembly  142 , in accordance with an embodiment of the present invention. 
         [0327]      FIG. 3C  shows a left side view of the reader optics assembly, in accordance with an embodiment of the present invention. 
         [0328]      FIG. 3D  is a forward scatter detection assembly  130 , in accordance with an embodiment of the present invention. This assembly contains LEDs,  352 , to illuminate a reading channel (such as reading channel  1452  ( FIG. 14M ) during an autofocus process, a stop  358 , to block low angle scatter and a lens  356  to collect the desired forward scatter for the detection photodiode. (forward scatter detector  130 ,  FIG. 1B ). 
         [0329]      FIG. 3E  is a side view of forward scatter detection assembly  130 , in accordance with an embodiment of the present invention. Shown in this view are an illumination lens  350 , collection lenses  356 ,  357 , and  358 , as well as a detection photodiode  360 . 
         [0330]      FIG. 4A  shows a cutaway view of reader assembly  130 , in accordance with an embodiment of the present invention. This is cutaway view of the reader assembly showing its components in its front and on a left side. These components include an ITX board  122 , a cartridge handling unit  128 , and the forward scatter detection assembly,  130 . 
         [0331]      FIG. 4B  shows an exploded right side view of a reader assembly  129 , in accordance with an embodiment of the present invention. The three major components in this view are a reader optics assembly  142 , a cartridge handling unit  128 , and a forward scatter detection module  130 . 
         [0332]      FIG. 4C  shows a left side blown up view of the reader assembly, in accordance with an embodiment of the present invention. Shown in this view are ITX computer board  122 , cartridge handling unit  128 , forward scatter detection assembly  130 , and the other side of the reader optics assembly,  142 . 
         [0333]      FIG. 4D  shows a rear view of cartridge handling unit (CHU)  128 , in accordance with an embodiment of the present invention. In this view, a handle  199  of the inserted cartridge,  110 , can be seen. Sensors  412  are configured therein to detect the position of motors  410 , and actuators  414 , which are adapted to crush the blisters, as well as an actuator  416  ( 940 ,  FIG. 9 ,  1415 ,  1417   FIGS. 14A-L ), can be seen on the shafts  417  of the motor. An opening  418  is provided for the microscope objective  438  to view the reading channel on the cartridge. 
         [0334]      FIG. 4E  shows a front view of a cartridge handling unit (CHU), in accordance with an embodiment of the present invention. This figure shows the front view of the cartridge handling unit (CHU)  128 . In this view, the handle in the upper portion of cartridge  110  can be seen. A port  420  to view the microfluidic path is provided. This port is viewed by a camera  430 , in order to ensure that the correct operation occurs within the cartridge. Another opening  441  is provided for the forward scatter to exit the cartridge handling unit and be observed by the forward scatter detection assembly  130 . 
         [0335]      FIG. 5  shows an exploded view of a reader assembly  130 , in accordance with an embodiment of the present invention. 
         [0336]      FIG. 6  is a simplified illustration of a disposable cartridge  6050  for rapid determination of a medical condition, in accordance with an embodiment of the present invention; 
         [0337]    Disposable cartridge  6050  is adapted to receive a bodily fluid, such as, but not limited to, blood, urine, serum or plasma. The disposable cartridge is constructed and configured to have several different sections  6052 ,  6054 ,  6056  and  6058 . Section  6052  is a body fluid aspiration section, which is adapted to receive the body fluid directly or indirectly from the patient (or animal) and this section acts as a reservoir of the body fluid. 
         [0338]    Disposable cartridge  6050  comprises fluid conveying means between the sections; such as, but not limited to, air pressure, liquid pressure, mechanical means and combinations thereof. Body fluid aspiration section  6052  is adapted to convey a predetermined quantity of the body fluid (a body fluid sample  6051 ) to a pre-analytical sample processing section  6054 . 
         [0339]    In pre-analytical sample processing section  6054 , at least one preparatory step is performed on the body fluid such as, but not limited to:
       a) incubation with at least one antibody;   b) incubation with at least one antigen;   c) staining of at least one cell type in the body fluid;   d) enzymatic lysing of at least one cell type of the body fluid;   e) osmotic lysing of at least one cell type of the body fluid;   f) heat or cool at least part of the bodily fluid;   g) addition of reference material to the bodily fluid; and   h) chemical reaction with at least one element of the body fluid.       
 
         [0348]    The pre-treated sample of bodily fluid is then conveyed from pre-analytical sample processing section  6054  to a sample excitation/interaction zone or section  6056 . This pre-treated sample may be conveyed continuously or in a batch mode to sample excitation/interaction section  6056 . 
         [0349]      FIG. 7A  is a simplified schematic illustration of an optical arrangement of a reader optics assembly  400 , in accordance with an embodiment of the present invention; 
         [0350]    A laser  440  or other appropriate light source provides a light beam  442 , which may be directed towards a plurality of optical elements, including a dichroic filter  443 , a beam splitter  444 , a focusing lens  445 , a pinhole  446  and a silicon reader unit  447 , for recording a signal from a beam  442  directed through the objective  438  towards a sample  450  and returned to the optical unit. Additional optical elements may include an optional attenuator  448 , a high-pass filter  449 , a focusing lens  451 , a slit  452 , a concave grating  453 , and a PMT array  454 . This arrangement of elements, representing an embodiment of the present invention, allows for generation of excitation light, focusing it on a sample, collecting reflected and emitted light signal resulting from the interaction of the excitation light and fluorophores in the sample and recording said returned light so as to determine fluorescence of sample in response to light illumination from laser  440 . 
         [0351]    With respect to  FIG. 7A , the laser illumination  442  is reflected by the dichroic filter  443  through the objective  438  and focused on the channel containing the flowing particles  458 . This illumination excites the fluorophores attached to the protein markers that are bound to the cells. 
         [0352]    The resulting fluorescent illumination is collected by the objective  438  and because of the longer wavelength of this emission passes through the dichroic filter  443  and is reflected by the beam splitter  444  through the high pass filter  449 . The high pass filter blocks any reflected laser illumination. The focusing lens  451  focuses the multi-wavelength emission illumination on the slit  452 . The concave grating  453  images the slit at multiple wavelengths on the elements of the PMT array  454 . This completes the process of creating a multispectral detection of the fluorescent emission. 
         [0353]    While most of the illumination collected by the objective is reflected by the beam splitter  444  a small fraction is allowed to pass through and is focused by focusing lens  445  through a pinhole  446  on the silicon reader unit  447 , which may be a single photodiode or a focal plane array such as CCD sensor. During the focusing operation best focus is achieved when the signal on this reader unit  447  is maximized. When this signal is maximized, the intensity of the signal on the PMT array  454  is also maximized. 
         [0354]    Reference is now made to  FIG. 7B , which is another simplified schematic illustration of optical arrangement  460  of a reader optics assembly  400  ( FIG. 7A ), in accordance with an embodiment of the present invention; 
         [0355]    With respect to  FIG. 7B , as in  FIG. 7A , the laser illumination is reflected by the dichroic filter  472  through the objective  476  and focused on the channel containing the flowing particles. This illumination excites the fluorophores attached to the protein markers that are bound to the cells. The resulting fluorescent illumination is collected by the objective  476  and because of the longer wavelength of this emission passes through the dichroic filter  472  and is reflected by the beam splitter  468  through the high pass filter  470 . The high pass filter  470  blocks any reflected laser illumination. The focusing lens  466  focuses the multi-wavelength emission illumination on the slit  478 . The concave grating  482  images the slit at multiple wavelengths on the elements of the PMT array  476 . This completes the process of creating a multispectral detection of the fluorescent emission. While most of the illumination collected by the objective  476  is reflected by the beam splitter  468  a small fraction is allowed to pass through and is focused through a pinhole  464  on the silicon reader unit  462 . During the focusing operation best focus is achieved when the signal on this reader unit is maximized. When this signal is maximized, the intensity of the signal on the PMT array  476  is also maximized. 
         [0356]      FIG. 8A  is a schematic representation of one example of multi-wavelength excitation in the optical unit of  FIG. 7A  or  7 B, in accordance with an embodiment of the present invention; 
         [0357]    Reference is now made to  FIG. 8A , which is a schematic representation  500  of one example of multi-wavelength excitation in the optical unit of  FIG. 7A  or  7 B, in accordance with an embodiment of the present invention.  FIGS. 8A-8C  show an extension of the optical configuration in  FIGS. 7A and 7B , to allow multiple excitation wavelengths. 
         [0358]      FIG. 8A  shows the configuration for combining multiple lasers of different wavelengths to yield a single coaxial beam  514  containing all of the wavelengths. Two different wavelengths, such as green  502  and red  506 , may be combined using a dichroic mirror  504 . One of the beams, red  506  is reflected by the dichroic mirror, while the second beam, green  502  passes through the dichroic mirror to yield a single beam  508 , yellow, containing both wavelengths. This combined wavelength beam is now used as one of the inputs to a second dichroic mirror  516  with the third wavelength  512  being reflected by the second dichroic mirror to yield a single coaxial beam  510  containing all three wavelengths. 
         [0359]    Reference is now made to  FIG. 8B , which shows a graphical output  520  of transmission as a function of wavelength for dichroic filter  500  of  FIG. 7B , employing the multi-wavelength excitation of  FIG. 8A , in accordance with an embodiment of the present invention. 
         [0360]    A multiband dichroic mirror (not shown) similar, or identical to, mirror  552  of  FIG. 8C  is used to illuminate the sample through an objective  554  ( FIG. 8C ), while allowing the resulting emission to pass through dichroic mirror  552  at all wavelengths, except those of multibeam excitation  514  ( FIG. 8A ). 
         [0361]    In this way the same epi-configuration used with a single wavelength can, in fact, be used with appropriate changes to dichroic mirror  552  and the addition of multiple lasers  502 ,  506 ,  512  to provide multi-wavelength excitation, while maintaining virtually all of the detection wavelengths of a single excitation system. 
         [0362]    Turning to  FIG. 8C , a schematic representation of part  550  of the optical unit is seen, employing multi-wavelength excitation of  FIG. 8A  and the dichroic filter of  FIG. 5B , in accordance with an embodiment of the present invention. Part  550  may, in some cases, replace subsystem  475  ( FIG. 7B ). 
         [0363]    Table 1 shows representative values for representative components for use in the present invention. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Laser Wavelength 
                 405 
                 nm 
                 488 
                 nm 
               
             
          
           
               
                 Laser Power 
                 50 
                 mW 
                 20 mW or 50 mW 
               
             
          
           
               
                 Sensing Spectral Range 
                 200 
                 nm 
                 200 
                 nm 
               
               
                 Spectral Resolution 
                 25 
                 nm 
                 25 
                 nm 
               
             
          
           
               
                 Number of Detectors 
                 8 
                 8 
               
               
                 Collecting Optics 
                 Microscope Objective 
                 Microscope Objective 
               
               
                   
                 N.A. &gt; 0.4, W.D. ≈ 
                 N.A. &gt; 0.4, W.D. ≈ 
               
               
                   
                 6 mm 
                 6 mm 
               
               
                 Detector Type 
                 S.S. PMT 8 ch 
                 S.S. PMT 8 ch 
               
               
                   
               
             
          
         
       
     
         [0364]    While much of the previous discussion has focused on the optical elements of some embodiments of the present invention, one of the key components of the diagnostic system herewith presented is a disposable sample cartridge. 
         [0365]    Reference is now made to  FIG. 9A , which is a schematic view of a sampling cartridge  110  of  FIG. 1A , in accordance with an embodiment of the present invention. The cartridge  650  includes a pre-analytical component  652  into which a sample (not shown) may be introduced. 
         [0366]    The sample will generally be blood, either whole or a component (serum, etc.) thereof. Other liquid samples may additionally or alternatively be employed. In the pre-analytical component  652 , the sample is allowed to interact with chemicals pre-packaged into component  652 . The interaction may be either passive or include active mixing. The chemicals included in the analytical component  652  may be either wet or dry, and generally include antibodies associated with fluorescent probes. Antibodies are pre-selected for their ability to bind with predetermined biological markers or the like. In a typical experiment, a predetermined volume (generally less than 50 microliters) of blood is introduced into the pre-analytical component  652  of a disposable cartridge  650 . The sample is actively mixed with chemical reagents present in the pre-analytical component  652  for a predetermined period of time, generally less than ten minutes. The sample is then moved through a capillary region  653  by means to be discussed, where it is exposed to a light beam  642  delivered from an objective  638 . Direction of sample flow is as shown by the arrow in the capillary region  653 . 
         [0367]    The capillary region  653  is designed to allow flow of particles in a single-file past the light beam  642 . Such an arrangement allows both for counting the number of particles as well as individual interrogation of particles to determine the presence of biological markers (via their associated fluorescent tags) on each particle. Such a physical arrangement allows for detection of one or more biological markers (independent of particle-specific properties such as size, shape, and number) on each particle. 
         [0368]    Finally, there is a collection component  654  which receives sample after exposure to light beam  642 . This is a waste region and allows for a completely self-contained disposable for sample preparation, analysis and waste collection. It is noted that the disposable cartridge may be of any relevant shape and is shown as it is in  FIG. 6  for ease of understanding of its components and functionality. 
         [0369]    As mentioned above, the sample, after pre-analytical treatment to allow for binding of fluorescent tag to cells/particles, must flow under a light beam  642 , produced by an optical unit (not shown). The flow is generally “single file” so as to allow for accurate determination of cell-specific markers on each analyzed cell. Methods to induce flow include but are not limited to electrical stimulation, chemical induction, and vacuum pull. In an electrical stimulation system, charge is applied across the capillary region  653  so as to induce charged particles to move from the pre-analytical component  652  towards the collection component  654 . The charge could be supplied by the cytometer in which the disposable cartridge  650  is placed or from an external source. 
         [0370]    Alternatively, the capillary region may include chemical features (hydrophilic/hydrophobic; positive/negative charge) to encourage sample to move from left to right as shown in  FIG. 9A . Alternatively, a vacuum from the collection component  654  could be applied to pull sample from the pre-analytical component  652  through the capillary region  653 . Other methods may be employed to get liquid sample to move underneath the light beam  642  for analysis. 
         [0371]    As described herein, the optics and sample handling have been handled separately. Such an arrangement is not mandatory, as some of the optical features needed for proper sample analysis may be included in a disposable cartridge. 
         [0372]    Reference is now made to  FIG. 9B , which shows a schematic view of disposable cartridge  800  in flow-cytometer device, such as system  100  in accordance with an embodiment of the present invention. Attention is currently turned to  FIG. 9B  which shows an expanded view of a capillary region  853 . 
         [0373]    In the capillary region  853 , particles flow in the direction as suggested by the arrow  880 . Particles  890  flow past an objective  838  that shines light  842  through the capillary  853 . Flow restriction elements  894  may be present in the capillary region  853  so as to encourage particles  890  to move past the light  842  in a nearly single-file manner. Passage of multiple particles together may be resolved through processing software. 
         [0374]    A molecular marker  895  on a particle  890  may be illuminated by light  842  and its fluorescence will be captured by a proximate photomultiplier tube  899 . The photomultiplier tube  899  may distinguish the wavelength of the fluorescence and thus which biological marker  895  is present on particle  890 . Thus, the systems of the present invention may determine which biological markers are present on particles  890 , which are detected in the systems of the present invention. A photomultiplier tube  899  may have a plurality of tubes or an array of elements for fine wavelength discrimination and alternatively may be replaced with film, CCD or other appropriate light-receiving reader unit. It should be understood that  FIG. 9B  shows one embodiment of the configuration of system  101  ( FIG. 1 ) in a transmissive configuration, wherein detector (photomultiplier tube  899 ) is disposed on an opposing side of the cartridge  800  to objective  838 . 
         [0375]    The systems of the present invention comprise controller software which are adapted to run a diagnostic process. It is understood that the controller software may be an integral part of the flow-cytometer or alternatively be installed on an associated computing device  122  ( FIG. 1B ), which may include, but not be limited to, a laptop computer, iPod, iPad, cell phone or mainframe computer. 
         [0376]    Reference is now made to  FIG. 10 , which is a simplified flowchart  1000  of a method for rapid determination of a medical condition, in accordance with an embodiment of the present invention. It is to be understood that the method described herein depicts one non-limiting embodiment of the present invention for determining the health state of a patient. Further embodiments are also construed to be part of the present invention. 
         [0377]    In a body fluid provision step  1002 , a body fluid, such as blood, urine, serum or plasma is provided from a human or animal patient. Typically, the sample is fresh, but may also be a stored, refrigerated or frozen-thawed sample. The fluid is typically liquid and at a temperature of 4-37° C. 
         [0378]    In a body fluid introduction step  1004 , part or all of the body fluid sample  6051  ( FIG. 6 ) is introduced into disposable cartridge ( 110 ,  FIG. 1A ). 
         [0379]    In a reacting step  1006 , the fluid sample is reacted with at least one reactant in the cartridge forming a treated sample. According to some embodiments, this step is performed in pre-analytical sample processing section  6054  ( FIG. 6 ) as described in detail hereinabove. 
         [0380]    In an impinging step  1008 , radiation is impinged on the treated sample, such as, but not limited to, in a sample excitation/interaction section  6056 , thereby generating a plurality of spectrally distinct signals in the direction of optics unit  142  ( FIG. 1C , see description hereinabove). 
         [0381]    In a spectral emissions detection step  1010 , a plurality of spectrally distinct signals is detected by multiple emission detector  454  ( FIG. 7A ). The detector outputs data. 
         [0382]    Thereafter, in a data processing step  1012 , the outputted data is processed by signal processor  6036  ( FIG. 6 ) and/or by computer  122  ( FIG. 1C ) to provide an output indicative of a medical condition. 
         [0383]      FIG. 11  is a three-dimensional graph showing the optical output over time of reference beads (RM) relative to a sample from a human patient (PMN), in accordance with an embodiment of the present invention. 
         [0384]      FIG. 11  shows a three-dimensional graph showing the optical output over time of reference beads (RM) relative to a sample from a human patient (PMN), in accordance with an embodiment of the present invention. The emission amplitude in the six bands, 500-525 nm, 525-550 nm, 550-575 nm, 575-600 nm, 600-625 nm and 625 to 650 nm is displayed in the graph for each sample time. Different fluorophores have different emission spectra. It can be appreciated that both spectral content or shape and amplitude at individual wavelengths are significantly different for neutrophils stained with Acridine Orange (AO) and reference beads (RM) containing a bright broad spectrum fluorophore. The peak of the AO emission is in the 525-550 nm band, while that of RM is in the 500-525 nm band and is of a significantly greater amplitude than AO in any band. 
         [0385]      FIGS. 12A-12C  show graphs of optical outputs over time of the reference beads and the sample from a human patient, in accordance with an embodiment of the present invention. 
         [0386]    Turning to  FIGS. 12A-12C , there can be seen graphs of optical outputs over time of the reference beads and the sample from a human patient, in accordance with an embodiment of the present invention. In these two-dimensional figures, the traces from each of the bands are overlaid on the same graph.  FIG. 12A  shows the boxed pulses from neutrophils in  FIG. 12B . It is clear from these graphs that the amplitude in the 525-550 nm channel exceeds the amplitude in the 500-525 nm channel, which is the characteristic of AO.  FIG. 12C  shows a comparison of the AO stained neutrophil emission spectrum to that of the RM emission spectrum. The relative amplitude of the spectrum in the 500-525 nm band to that of the amplitude in the 525-550 nm band clearly distinguishes the two fluorophores. In addition, the maximum amplitude of the RM emission is significantly greater than that of AO. 
         [0387]    The systems of the present invention, as described and shown herein provide uses, such as, but not limited to, at least one of the four following scenarios:
       a) When multiple pieces of information, such as biological markers and white cell state are required in order to make an accurate diagnostic determination;   b) When multiple sequential measurements must be made in order to determine the position of a patient on an illness curve;   c) When white cell and similar data are needed quickly and in a POC environment; and   d) When fluorescent signals overlap in wavelength and there is need to determine relative contribution of each signal for a given wavelength range.       
 
         [0392]    The instant invention includes software and algorithms for proper data analysis and conversion of raw fluorescence data into actual concentrations of relative biological markers. 
         [0393]      FIG. 13A  is an outer side view of a cartridge assembly  1300 , in accordance with an embodiment of the present invention and  FIG. 13B  shows an inner side view  1350  of a cartridge assembly  1300 , in accordance with an embodiment of the present invention. 
         [0394]      FIG. 14A-14O  show a sequence of process events in a cartridge assembly, in accordance with an embodiment of the present invention; 
         [0395]      FIG. 14A-14O  are a sequential set of schematic drawings of the operation of a system  101  ( FIG. 1A ) for detecting a biological condition, in accordance with an embodiment of the present invention. 
         [0396]    In  FIG. 14A , a blood sample  1401  enters a specimen receiving element  1418  and fills a chamber  1404 . 
         [0397]    In  FIG. 14B , a blister  1420  comprising a treatment composition  120  ( FIG. 1 ) is pressed and an antibody cocktail is mixed with 10 microliters of the blood sample. 
         [0398]    In  FIG. 14C , a mixing bellows  1415  is pressed and this effects mixing of the antibody cocktail and the 10 microliters of the blood sample in a first mixing chamber  1412  to form a first mixture  1403 . 
         [0399]    In  FIG. 14D , the bellows is released and mixture  1403  is siphoned along a tortuous channel  1413  and into a second mixing chamber  1411 . 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. 
         [0400]    In  FIGS. 14E-14G , a second composition blister  1422  is pressed, releasing a second composition  122  ( FIG. 1 ), such as a lysis composition thereby forming a second mixture  1405 . The second mixture is mixed by pressing of bellows  1415 , the second mixture returns from the second mixing chamber, back along tortuous channel  1413  to the first mixing chamber. Every time the bellows is pressed the mixture moves towards the second chamber  1411  and every time it is released, it returns, wholly or in part to the first chamber  1412 . This mixing may be performed multiple times. 
         [0401]    In  FIGS. 14H-14J , a third blister  1424  is released comprising a third composition  124  ( FIG. 1 ), such as a control reference, into the second mixing chamber, thereby forming a third composition  1407 . The third mixture is mixed by pressing of bellows  1415 , the third mixture returns from the second mixing chamber, back along tortuous channel  1413  to the first mixing chamber. Every time the bellows is pressed the mixture moves towards the second chamber  1411  and every time it is released, it returns, wholly or in part to the first chamber  1412 . This mixing may be performed multiple times. 
         [0402]    In  FIGS. 14J-14M , a reading bellows  1417  is pressed, which forces some of the third composition towards a reading cuvette  1430 . 
         [0403]    In  FIGS. 14N-14O , particles  1460  from the third composition flow from the cuvette  1430  along a reading channel  1452  to a reading region  1450 . The cells pass through the reading region and are excited by one or more lasers  1462 ,  1463 . At least one excitation laser beam  1464  impinges on cell  1460  and an emission beam  1466  is detected by a detector  1470 . In one example, this is cell emission fluorescence and detector  1470  is a spectrometer. 
         [0404]      FIG. 15  is a schematic illustration of a micro flow spectrometer reading, in accordance with an embodiment of the present invention; 
         [0405]    An individual cell  1505  flows through a detection region  1510  in a microfluidic channel (seen as  1452 ,  FIG. 14M ). Additionally, tagged cells  1520  labeled with antibodies conjugated with multiple wavelength fluorescent tags flow through the detection region. A diode laser  1530  impinges a ray/beam  1510  onto the cells and tagged cells. The cells and tagged cells emit different emission spectra (not shown). An optical grating  1540  disperses emission spectra via a grating  1540  into its constituent wavelengths  1550 . 
         [0406]    A photomultiplier tube (PMT) array  1560  or avalanche diode array detects fluorescence at 8 different spatial locations corresponding to 8 spectral regions. 
         [0407]      FIG. 16  is a flow chart of a method for optical processing  1600 , in accordance with an embodiment of the present invention. 
         [0408]    In a forming laser step  1602 , a laser excitation beam shape is formed. 
         [0409]    The excitation beam is reflected from a dichroic mirror  504  ( FIG. 8A , or  472   FIG. 7B ) and through objective  476  ( FIG. 7B ) onto a reading channel  1452  ( FIG. 14M ), in a reflecting step  1604 . 
         [0410]    In a forward scatter measuring step  1606 , the forward scatter from particles  1460  ( FIG. 14N ) in the reading channel is measured to detect events. 
         [0411]    Thereafter, in a passing step  1608 , particle fluorescent emission is allowed to pass through a dichroic mirror and be reflected from a beamsplitter  468  into a detection path. 
         [0412]    In an imaging step  1610 , parts of the beam emission, which are not reflected are passed through the beamsplitter onto an image sensor, such as silicon detector  462  ( FIG. 7B ). 
         [0413]    In parallel to step  1610 , the reflected part of the beam is filtered in a beam filtering step  1612  in the detection path to allow only wavelengths above an excitation wavelength to pass through a filter. 
         [0414]    In a focusing step  1614 , the filtered beam from step  1612  is focused onto a pinhole or slit to select a reading zone region to be analyzed. 
         [0415]    Thereafter, in a dispersing step  1616 , the dispersed pinhole or slit is dispersed and imaged onto a multi-element electrooptical detector ( 6034 ,  FIG. 6 ). 
         [0416]      FIGS. 17A-17B  are schematic illustrations of steps of use of a graphical user interface, in accordance with an embodiment of the present invention; 
         [0417]    Upon powering up the unit a first screen  1702  appears with a message notifying the user that the system is performing a self-check along with a countdown indicator  1703 . Once the self-check is complete, an assay selection screen  1704  appears. The user touches the button corresponding to the assay to be performed. The next screen  1706  is used to enter the patient identification. This may be done by touching the numerals of the touchpad  1709  or by scanning a barcode. Once the entry is complete, the user touches the forward button  1707  and a screen requesting the user to enter the cartridge  1708  appears. Once the user inserts the cartridge the system checks to ensure that the cartridge identified by its barcode label corresponds to the selected assay and begins the processing. While processing, a screen  1710  is displayed showing the processing progress and the time remaining. Once the pre-analytical and analytical processing is completed the results are displayed on a screen  1712  with an indication of where the results lie in the range of possible results  1713 . After the user touches a “proceed to next screen indicator”  1711  a screen instructing the user to remove the cartridge  1714  appears. The user has the option of repeating this test with another sample by pressing the repeat icon  1715  or displaying the most recent results on a screen  1716 . 
         [0418]    Reference is now made to  FIG. 18 , which is a simplified illustration of a method for a disposable cartridge  1850  of system  101  of  FIG. 1A  for rapid determination of a medical condition, in accordance with an embodiment of the present invention. 
         [0419]    When practicing the method of disposable cartridge  1850  a bodily fluid, such as, but not limited to, blood, urine, serum or plasma is transferred from the donor to the cartridge  1851 . The disposable cartridge method includes multiple steps to effect the analysis and diagnosis  1852 ,  1854 ,  1856  and  1858 . In step  1852  a body fluid aspiration step, receives the body fluid directly or indirectly from the patient (or animal) and transfers the body fluid to a reservoir. 
         [0420]    The disposable cartridge method  1850  utilizes fluid conveying means, such as, but not limited to, air pressure, liquid pressure, mechanical means and combinations thereof to move fluids. Body fluid aspiration step  1852  is adapted to convey a predetermined quantity of the body fluid (a body fluid sample  1851 ) for a pre-analytical sample processing step  1854 . 
         [0421]    In pre-analytical sample processing  1854 , at least one preparatory step is performed on the body fluid such as, but not limited to:
       i) incubation with at least one antibody;   j) incubation with at least one antigen;   k) staining of at least one cell type in the body fluid;   l) enzymatic lysing of at least one cell type of the body fluid;   m) osmotic lysing of at least one cell type of the body fluid;   n) heating or cooling at least part of the bodily fluid;   o) addition of reference material to the bodily fluid; and   p) chemical reaction with at least one element of the body fluid.       
 
         [0430]    The pre-treated sample of bodily fluid is then transferred ( 1855 ) after pre-analytical sample processing step  1854  to a sample excitation/interaction step  1856 . This pre-treated sample transfer for excitation/interaction  1856  may be performed continuously or in a batch mode. 
         [0431]    Part of sample excitation/interaction  1856  is to position the sample to sit in the light path of an excitation illumination. The excitation illumination passes radiation, such as coherent or incoherent radiation in or outside the visible range into the pre-treated sample. Resultant emission or emissions from the pre-treated sample is detected  1834 , and processed  1836  to produce a report  1812  summarizing the analysis and diagnosis. 
         [0432]    Multi-spectral emission detection  1834  receives the emission from the pre-treated sample in multiple spectral bands. In some cases these bands are non-overlapping bands. Multi-spectral emission detection  1834  is adapted to pass data representing the spectral bands to multi-spectral fluorescence signal processing  1836 . 
         [0433]    Multi-spectral fluorescence signal processing  1836  may comprise two or more sub-elements (not shown) including:
       a) a photon counting analysis;   b) other detecting analysis elements (not shown) for measuring other optical outputs of multi-spectral emission detection  1834 .       
 
         [0436]    The method further comprises a spent sample disposal method  1858 , for receiving a sample from the sample excitation/interaction processing. 
         [0437]    The method further comprises computer program  1810 , the computer program is adapted to receive data related to the plurality of spectrally distinct signals and a processor, adapted to process said data and to output at least one output related to said medical condition. One type of output provided is a visual output which is outputted onto a screen  1812  of the computer. 
         [0438]      FIG. 19A  is a simplified flow chart of a method  600  for differentiating between different particles, in accordance with an embodiment of the present invention. 
         [0439]    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 crosscorrelations  606 . One or more of these derived time series may be used to determine event locations. 
         [0440]    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. 
         [0441]    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 . 
         [0442]    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. 
         [0443]      FIG. 19B  is a flow chart of an algorithm for biological detection  1900 , in accordance with an embodiment of the present invention; 
         [0444]      FIG. 19  B shows schematically that the CD4/CD8 assay is performed by determining a particle type for each event in the event store  1912 . One method to accomplish this particle selection is to use K means clustering to determine data clusters in the event store based on the signatures Alexa 488, PE 488N and PEAF 488N as shown in  FIG. 25 . Based on these three signatures events with large values of PE 488N are classified as CD4 positive lymphocytes  1938  since the phycoerythrin (PE) fluor is attached to the CD4 antibody. Events with large values of Alexa 488 are classified as CD8 positive lymphocytes  1940  since the Alexa 488 fluor is attached to this CD8 antibody. Events with large values of PEAF 488N are classified as lymphocytes since this fluor is attached to the CD3 antibody which is expressed by lymphocytes to the exclusion of other WBC. The group  1942  has large values of PEAF 488N but small values of PE 488N and Alexa 488 which corresponds to lymphocytes not expressing either CD4 or CD8. Finally, non-lymphocytes WBC  1936  may be determined by a pan WBC antibody for those events not expressing CD3, and rejects as those events not expressing the pan WBC antibody. 
         [0445]      FIGS. 20A-20B  shows bandwidth leveled and smoothed arrays, in accordance with an embodiment of the present invention. 
         [0446]      FIGS. 20A and 20B  show the typical response of the system to fluorescent beads with an F488 signature. The response in 25 nm bands from 500 nm to 700 nm are respectively  2010 ,  2020 ,  2030 ,  2040 ,  2050 ,  2060 ,  2070 , and  2080 . The traces represent the outputs from the eight channel fluorescent detector with noise, characterized as the median trace value, subtracted. The large signals above the background level are the raw signal smoothed by a box car averager of length 10. The peak value for the F488 signature occurs in the range 525 to 549 nm,  2020 . The next highest value occurs in the range 500 to 524 nm,  2010 , with decreasing amplitudes in each 25 nm band,  2030 ,  2040 ,  2050 ,  2060 ,  2070 , and  2080 . 
         [0447]      FIGS. 21A-21B  are schematics for solving a fluor decomposition of an observed signal, in accordance with an embodiment of the present invention. 
         [0448]      FIG. 21A  is the Matlab function used to solve the matrix equation Ax=b for the signature value vector or vectors b corresponding to the observed eight channel emission values x. The \ function in Matlab is used to solve for x using the Matlab expression x=A\b. 
         [0449]      FIG. 21  B is a table of fluor signatures use as the matrix A described in FIG.  21 A. 
         [0450]      FIGS. 22A-22B  is a graphical comparison of system performance with FITC beads with MESF detection versus FACS, in accordance with an embodiment of the present invention. 
         [0451]      FIG. 22A  shows a comparison of the linearity of the instant invention with that of a Becton Dickinson FACS flow cytometer. The tabulated median values for the FITC MESF beads are shown in column  2211  in table  2210 . The median fluorescent intensity levels (in arbitrary units) for the FACS flow cytometer are shown in column  2212 , while those for the instant invention are shown in column  2213 . Column  2214  shows the number of events on which the median value is based for the instant invention. The linearity plot for the full range of values for the instant invention is shown in  2220  while that for the FACS flow cytometer is shown in  2230 . Also shown in each of these figures is the best fit line through the points along with the square of the correlation value, R 2 . Comparison of these plots shows comparable performance of the instant invention and the FACS flow cytometer. 
         [0452]      FIG. 22B  shows a comparison of the linearity of the instant invention  2240  with that of the FACS flow cytometer  2250  over the range restricted to the first four smallest data points. Again, comparable performance is demonstrated. 
         [0453]      FIGS. 23A-23B  show graphical displays of linearity of system performance with Alexa 488 MESF, in accordance with an embodiment of the present invention. 
         [0454]      FIGS. 23A and 23B  shows the linearity performance of the instant invention for the Alexa Fluor 488 MESF bead series when the system is run both add fast speed  2310  and flow speed  2330 . In this case the measure of performance is the F488 signature normalized to the length of the event. This normalized signature is designated F488N. The tabular listings  2320  and  2340  summarize the statistics for the regression line fit. 
         [0455]      FIG. 24  is a three-dimensional graph showing the optical output over time of a CD4-CD8 assay, in accordance with an embodiment of the present invention. 
         [0456]      FIG. 24  is a surface plot showing the relative amplitude of event emission in each of the detectors for a thirty second interval. The scale running from left to right  2440  is in 10 μs intervals. The scale running from right to left  2450  and numbered one through eight are the detector elements. Eight corresponds to waveband 1, seven corresponds to waveband 2, and finally one corresponds to waveband 8. An example of CD4 events tagged with phycoerythrin shown in  2510  in  FIG. 25  is the trace  2430 . An example of CD8 events tagged with Alexa Fluor 488 shown in  2520  in  FIG. 25  is the trace  2410 . Finally an example of non-CD4 non-CD8 lymphocytes tagged only with Alexa Fluor 610 shown in the group  2530  in  FIG. 25  is the trace  2420 . 
         [0457]      FIG. 25  is a graphical display showing a cluster analysis of a CD4-CD8 assay, in accordance with an embodiment of the present invention; 
         [0458]      FIG. 25  is the scatterplot matrix showing the result of applying K means clustering to the signatures Alexa 488N, PE488N and PEAF488N. The meaning of each of the clusters  2510 ,  2520  and  2530  is described in the description of  FIG. 19  B. 
         [0459]      FIGS. 26-27  are graphical displays showing cluster separations of the cluster analysis of  FIG. 19A , in accordance with an embodiment of the present invention. 
         [0460]      FIG. 26  is a scatterplot matrix showing four-color separation of the neutrophil, monocyte, lymphocyte and reference bead populations required to effect a CD64 assay. The leftmost column  2710  shows the complete 4-color separation. The top frame  2720  shows separation of NE &amp; LY from MO and BEADS based on Waveband2 (Alexa488) and separation of MO from BEADS based on Waveband4 (PE). 
         [0461]    The middle frame  2730  shows separation of LY from NE based on Waveband6 (A610). 
         [0462]    The bottom frame  2740  shows separation of beads from cells based on Waveband8 (Starfire Red). 
         [0463]    Since the separation is based on individual narrow bands (not signatures) 45 degree clusters  2750 ,  2760 ,  2770  show emission presence in two bands, which in each case is as expected as can be seen from the emission signatures in the table below. 
         [0464]      FIG. 28  is a comparison table of different array options, in accordance with an embodiment of the present invention. 
         [0465]      FIG. 28  is a tabular listing of photomultiplier arrays produced by Hamamatsu Corporation. The H95308 channel array is the one used in the current implementation of the extant invention. One skilled in the art will appreciate that finer resolution or greater extent of the spectral sampling can be achieved by using either the 16 or 32 channel array products. 
         [0466]      FIG. 29A  is a flowchart of a specific implementation of an algorithm  1300  for selecting groups of data from a scatterplot, in accordance with an embodiment of the present invention; 
         [0467]    The algorithm in  FIG. 29A  is a specific implementation of the general algorithm in  FIG. 29B  to select each of the groups  3010 ,  3020 ,  3030  and  3040  ( FIG. 30 ) and determine specific parameter values in each of the groups. 
         [0468]    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 . 
         [0469]    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. 31A . The next step is to remove this group from the overall dataset as shown in  FIG. 29A  Step  1322 . The removed group is the bead dataset  1324 . 
         [0470]    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. 
         [0471]    In an analyzing PE histogram set step,  1344 , the data is manipulated to find a group corresponding to monocytes. 
         [0472]    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 (full name PEAF®488) (see above for beads and PE) signature in an order according to PEAF signature ordering step  1362 . 
         [0473]    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. 
         [0474]    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 . 
         [0475]    In an order by Diode1 signature step  1382 , dataset  1380  is analyzed according to a Diode1 signature (see above). Dataset  1380  is then analyzed in an analyzing step  1384  to find a group of data having properties of neutrophils. 
         [0476]    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. 
         [0477]    Reference is now made to  FIG. 29B , 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. 
         [0478]    In a first ordering signature step  1305  a first signature is used to order the dataset of waveband and signature values  1303 . 
         [0479]    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. 31A  to select the group  3010 . This is a small group  1404  at the upper end of group  1402  in the histogram  1400  in  FIG. 31A . 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. 31B  Step  1323 . A removed group is a 1st signature dataset  1325 . 
         [0480]    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. 
         [0481]    In an analyzing 2 nd  signature histogram set step,  1345 , the data is manipulated to find a group corresponding to the 2 nd  signature. 
         [0482]    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 . 
         [0483]    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. 
         [0484]    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  a signature groups removed  1381 . 
         [0485]    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. 
         [0486]    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 group) is used in a computing step  1393  to compute desired metric from the group parameters. 
         [0487]      FIG. 31A  is a histogram  1400  of data of Starfire Red (SFR) signature values, in accordance with an embodiment of the present invention. 
         [0488]      FIG. 31B  shows a plot  1450  of a polynomial  1452  and first derivative thereof  1456  and second derivative thereof  1458  of histogram  1400  shown in  FIG. 31A , in accordance with an embodiment of the present invention. 
         [0489]    Referring to  FIG. 31B , the method of determining an upper group  1404  in  FIG. 31A  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. 31A ). 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 . 
         [0490]      FIG. 32A  is a histogram  1500  of data of PE488 signature values, in accordance with an embodiment of the present invention. 
         [0491]      FIG. 32B  shows a polynomial fitted to the histogram in  FIG. 32A  as well as a corresponding first derivative  1556  and a second derivative  1558 , in accordance with an embodiment of the present invention. 
         [0492]    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. 32A . 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. 32B  shows the polynomial  1552  fitted to data  1554  of histogram  1500  in  FIG. 32A  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. 
         [0493]    As noted in  FIG. 31A , the monocyte group  1504  is removed from the dataset which now contains primarily lymphocytes, neutrophils and other particles such as unlysed erythrocytes and other debris. 
         [0494]      FIG. 33A  is a histogram  1600  of data  1602  of PEAF488 signature values, in accordance with an embodiment of the present invention. 
         [0495]      FIG. 33B  shows a polynomial  1652  fitted to histogram data  1654  from  FIG. 33A  as well as a corresponding first derivative  1656  and a second derivative  1658 , in accordance with an embodiment of the present invention. 
         [0496]    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. 33A  and the corresponding polynomial fit with its first and second derivatives are shown in  FIG. 33B . 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. 29A  leaving a dataset  1380  which now contains primarily neutrophils and other particles such as unlysed erythrocytes and other debris. 
         [0497]    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 Diode1 (forward scatter detector) channel. A histogram of the Diode1 channel is shown in  FIG. 34A . 
         [0498]      FIG. 34A  is a histogram  1700  of data of Diode 1 channel signature values, in accordance with an embodiment of the present invention. 
         [0499]      FIG. 34B  shows a polynomial  1752  fitted to data  1754  from the histogram in  FIG. 34A , as well as a corresponding first derivative  1756  and a second derivative  1758 , in accordance with an embodiment of the present invention. 
         [0500]    As described above, an upper group  1704  ( FIG. 34A ) 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. 29A . 
         [0501]    Within each group various parameters may be computed from the fields in the dataset. An example is shown in the following table. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Observations 
                 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 
               
               
                   
               
             
          
         
       
     
         [0502]    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. 
         [0503]    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  FIG. 29B . 
         [0504]    Other suitable operations or sets of operations may be used in accordance with some embodiments. Some operations or sets of operations may be repeated, for example, substantially continuously, for a pre-defined number of iterations, or until one or more conditions are met. In some embodiments, some operations may be performed in parallel, in sequence, or in other suitable orders of execution. 
         [0505]    Discussions herein utilizing terms such as, for example, “processing,” “computing,” “calculating,” “determining,” “establishing”, “analyzing”, “checking”, or the like, may refer to operation(s) and/or process(es) of a computer, a computing platform, a computing system, or other electronic computing device, that manipulate and/or transform data represented as physical (e.g., electronic) quantities within the computer&#39;s registers and/or memories into other data similarly represented as physical quantities within the computer&#39;s registers and/or memories or other information storage medium that may store instructions to perform operations and/or processes. 
         [0506]    Some embodiments may take the form of an entirely hardware embodiment, an entirely software embodiment, or an embodiment including both hardware and software elements. Some embodiments may be implemented in software, which includes but is not limited to firmware, resident software, microcode, or the like. 
         [0507]    Some embodiments may utilize client/server architecture, publisher/subscriber architecture, fully centralized architecture, partially centralized architecture, fully distributed architecture, partially distributed architecture, scalable Peer to Peer (P2P) architecture, or other suitable architectures or combinations thereof. 
         [0508]    Some embodiments may take the form of a computer program product accessible from a computer-usable or computer-readable medium providing program code for use by or in connection with a computer or any instruction execution system. For example, a computer-usable or computer-readable medium may be or may include any apparatus that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device. 
         [0509]    In some embodiments, the medium may be or may include an electronic, magnetic, optical, electromagnetic, InfraRed (IR), or semiconductor system (or apparatus or device) or a propagation medium. Some demonstrative examples of a computer-readable medium may include a semiconductor or solid state memory, magnetic tape, a removable computer diskette, a Random Access Memory (RAM), a Read-Only Memory (ROM), a rigid magnetic disk, an optical disk, or the like. Some demonstrative examples of optical disks include Compact Disk-Read-Only Memory (CD-ROM), Compact Disk-Read/Write (CD-R/W), DVD, or the like. 
         [0510]    In some embodiments, a data processing system suitable for storing and/or executing program code may include at least one processor coupled directly or indirectly to memory elements, for example, through a system bus. The memory elements may include, for example, local memory employed during actual execution of the program code, bulk storage, and cache memories which may provide temporary storage of at least some program code in order to reduce the number of times code must be retrieved from bulk storage during execution. 
         [0511]    In some embodiments, input/output or I/O devices (including but not limited to keyboards, displays, pointing devices, etc.) may be coupled to the system either directly or through intervening I/O controllers. In some embodiments, network adapters may be coupled to the system to enable the data processing system to become coupled to other data processing systems or remote printers or storage devices, for example, through intervening private or public networks. In some embodiments, modems, cable modems and Ethernet cards are demonstrative examples of types of network adapters. Other suitable components may be used. 
         [0512]    Some embodiments may be implemented by software, by hardware, or by any combination of software and/or hardware as may be suitable for specific applications or in accordance with specific design requirements. Some embodiments may include units and/or sub-units, which may be separate of each other or combined together, in whole or in part, and may be implemented using specific, multi-purpose or general processors or controllers. Some embodiments may include buffers, registers, stacks, storage units and/or memory units, for temporary or long-term storage of data or in order to facilitate the operation of particular implementations. 
         [0513]    Some embodiments may be implemented, for example, using a machine-readable medium or article which may store an instruction or a set of instructions that, if executed by a machine, cause the machine to perform a method and/or operations described herein. Such machine may include, for example, any suitable processing platform, computing platform, computing device, processing device, electronic device, electronic system, computing system, processing system, computer, processor, or the like, and may be implemented using any suitable combination of hardware and/or software. The machine-readable medium or article may include, for example, any suitable type of memory unit, memory device, memory article, memory medium, storage device, storage article, storage medium and/or storage unit; for example, memory, removable or non-removable media, erasable or non-erasable media, writeable or re-writeable media, digital or analog media, hard disk drive, floppy disk, Compact Disk Read Only Memory (CD-ROM), Compact Disk Recordable (CD-R), Compact Disk Re-Writeable (CD-RW), optical disk, magnetic media, various types of Digital Versatile Disks (DVDs), a tape, a cassette, or the like. The instructions may include any suitable type of code, for example, source code, compiled code, interpreted code, executable code, static code, dynamic code, or the like, and may be implemented using any suitable high-level, low-level, object-oriented, visual, compiled and/or interpreted programming language, e.g., C, C++, Java, BASIC, Pascal, Fortran, Cobol, assembly language, machine code, or the like. 
         [0514]    Functions, operations, components and/or features described herein with reference to one or more embodiments, may be combined with, or may be utilized in combination with, one or more other functions, operations, components and/or features described herein with reference to one or more other embodiments, or vice versa. 
         [0515]    Any combination of one or more computer usable or computer readable medium(s) may be utilized. The computer-usable or computer-readable medium may be, for example but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, device, or propagation medium. More specific examples (a non-exhaustive list) of the computer-readable medium would include the following: an electrical connection having one or more wires, a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), an optical fiber, a portable compact disc read-only memory (CDROM), an optical storage device, a transmission media such as those supporting the Internet or an intranet, or a magnetic storage device. Note that the computer-usable or computer-readable medium could even be paper or another suitable medium upon which the program is printed, as the program can be electronically captured, via, for instance, optical scanning of the paper or other medium, then compiled, interpreted, or otherwise processed in a suitable manner, if necessary, and then stored in a computer memory. In the context of this document, a computer-usable or computer-readable medium may be any medium that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device. The computer-usable medium may include a propagated data signal with the computer-usable program code embodied therewith, either in baseband or as part of a carrier wave. The computer usable program code may be transmitted using any appropriate medium, including but not limited to wireless, wireline, optical fiber cable, RF, etc. 
         [0516]    Computer program code for carrying out operations of the present invention may be written in any combination of one or more programming languages, including an object oriented programming language such as Java, Smalltalk, C++ or the like and conventional procedural programming languages, such as the “C” programming language or similar programming languages. The program code may execute entirely on the user&#39;s computer, partly on the user&#39;s computer, as a stand-alone software package, partly on the user&#39;s computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user&#39;s computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider). 
         [0517]    The present invention is described herein with reference to flow chart illustrations and/or block diagrams of methods, apparatus (systems) and computer program products according to embodiments of the invention. It will be understood that each block of the flow chart illustrations and/or block diagrams, and combinations of blocks in the flow chart illustrations and/or block diagrams, can be implemented by computer program instructions. These computer program instructions may be provided to a processor of a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions/acts specified in the flowchart and/or block diagram block or blocks. 
         [0518]    These computer program instructions may also be stored in a computer-readable medium that can direct a computer or other programmable data processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable medium produce an article of manufacture including instruction means which implement the function/act specified in the flow charts and/or block diagram block or blocks. 
         [0519]    The computer program instructions may also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer implemented process such that the instructions which execute on the computer or other programmable apparatus provide processes for implementing the functions/acts specified in the flow charts and/or block diagram block or blocks. 
         [0520]    The flow charts and block diagrams in the Figures illustrate the architecture, functionality, and operation of possible implementations of systems, methods and computer program products according to various embodiments of the present invention. In this regard, each block in the flow charts or block diagrams may represent a module, segment, or portion of code, which comprises one or more executable instructions for implementing the specified logical function(s). It should also be noted that, in some alternative implementations, the functions noted in the block may occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and/or flow chart illustrations, and combinations of blocks in the block diagrams and/or flow chart illustrations, can be implemented by special purpose hardware-based systems that perform the specified functions or acts, or combinations of special purpose hardware and computer instructions. 
         [0521]    Although the embodiments described above mainly address assessing test coverage of software code that subsequently executes on a suitable processor, the methods and systems described herein can also be used for assessing test coverage of firmware code. The firmware code may be written in any suitable language, such as in C. In the context of the present patent application and in the claims, such code is also regarded as a sort of software code. 
         [0522]    The cartridges of the present invention 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.       
 
         [0529]    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. 
         [0530]    Table 2 shows some representative applications of apparatus  100  and methods of the present invention. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Applications of the apparatus and methods of this invention. 
               
             
          
           
               
                   
                   
                   
                 Typical Prior 
                 This 
                   
               
               
                   
                   
                 Relevant 
                 Art Laboratory 
                 invention 
               
               
                   
                   
                 FIGS. in 
                 Turnaround 
                 Turnaround 
               
               
                   
                 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, B H 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 
                 Cristofanilliet 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 3-5D 
                   
                 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 - CytoImmun - 
                   
                   
                 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 
               
               
                   
               
             
          
         
       
     
         [0531]    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. 
         [0532]    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. 
         [0533]    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. 
         [0534]    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. 
         [0535]    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. 
         [0536]    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. 
         [0537]    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. 
         [0538]    The sequence of transfer of the various treatment compositions may be important to the reaction sequence and is typically predefined. 
         [0539]    The reading region  1450  ( FIG. 14M-N ) is configured and constructed for one or more evaluation steps. 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.       
 
         [0550]    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. 
         [0551]    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 system  101 . 
         [0552]    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. 
         [0553]    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. 
         [0554]    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. 
         [0555]    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. 
         [0556]    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. 
         [0557]    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. 
         [0558]    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. 
         [0559]    The time required to complete an assay using system  101  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. 
       Example 
     Application No. 1 
     CD64 Infection &amp; Sepsis 
       [0560]    A cartridge  110  ( FIG. 1A ) 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). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Time sequences of steps in the methodology of the present 
               
               
                 invention for detecting sepsis using CD64 and CD163 antibodies. 
               
               
                 LeukoDx device- present invention 
               
             
          
           
               
                   
                   
                 Volume 
                 Duration 
                   
               
               
                 Step 
                 Description 
                 (uL) 
                 (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 
               
               
                   
               
             
          
         
       
     
         [0561]    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. 
         [0562]    According to some embodiments, the readout may comprise an optoelectronics core, which enables identification and detection of fluorescent signals. 
         [0563]    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. 
         [0564]    The contents of these publications are incorporated by reference herein where appropriate for teachings of additional or alternative details, features and/or technical background. 
         [0565]    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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