US20140170678A1 - Kits, compositions and methods for detecting a biological condition - Google Patents

Abstract

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

Description

CROSS REFERENCE TO RELATED APPLICATIONS
• The disclosures of the co-pending US Provisional Patent Application to Kasdan, et al, filed on Nov. 17, 2012, and titled “Kits, Compositions and Methods for Detecting a Biological Condition” and the co-pending US Provisional Patent Application to Kasdan, et al, filed on Nov. 17, 2012, and titled “Kits, Compositions and Methods for Rapid Chemical Detection” are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
• 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
• There are numerous medical conditions which are hard to diagnose. Often diagnosis by a physician is based on the physician's observation of combinations of symptoms in a patient. This sometimes leads to misdiagnosis. Furthermore, the patient's response to a treatment, whether drug or other modality is often followed up by physician's observation.
• 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.
• 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
• 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.
• In some embodiments of the present invention, improved methods, apparatus and kits are provided for detecting and diagnosing a biological condition in a patient.
• 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.
• 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.
• There is thus provided according to an embodiment of the present invention, a kit for evaluating a biological condition in a patient, the kit comprising;
• • a) a disposable element for receiving a biological specimen and for combining said specimen with at least one composition;
  • b) at least one composition comprising at least one detector moiety adapted to react with said specimen to form a reaction product, when said patient has said biological condition; and
  • c) at least one reporter element adapted to provide an indication of reaction product thereby providing the indication of the biological condition.
• Additionally, according to an embodiment of the present invention, the kit further comprises;
• • d) instructions for using the kit.
• Furthermore, according to an embodiment of the present invention, the disposable element is a disposable cartridge.
• Moreover, according to an embodiment of the present invention, the disposable cartridge is a disposable microfluidics cartridge.
• Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least one of the following elements:
• • a) a reservoir;
  • b) a pump;
  • c) a valve;
  • d) a conduit;
  • e) a motor;
  • f) a miniaturized flow cell;
  • g) a transport channel;
  • h) a microfluidic element;
  • i) a compressed gas holding element;
  • j) a compressed gas releasing element;
  • k) a nozzle element;
  • l) a mixing element;
  • m) a bellows element;
  • n) software adapted to activate said elements according to a specific sequence; and
  • o) hardware to activate said elements according to a specific sequence.
• Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least two of the elements.
• Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least three of the elements.
• Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least four of the elements.
• Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least five of the elements.
• Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least ten of the elements.
• Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least twenty of the elements.
• Additionally, according to an embodiment of the present invention, the disposable microfluidics cartridge comprises at least thirty of the elements.
• According to an embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with one hour.
• According to another embodiment of the present invention, the micro fluidics kit is configured to provide the rapid indication with thirty minutes.
• According to another embodiment of the present invention, the micro fluidics kit is configured to provide the rapid indication with fifteen minutes.
• According to another embodiment of the present invention, the micro fluidics kit is configured to provide the rapid indication with ten minutes.
• According to another embodiment of the present invention, the micro fluidics kit is configured to provide the rapid indication with five minutes.
• According to another embodiment of the present invention, the micro fluidics kit is configured to provide the rapid indication with one minute.
• According to another embodiment of the present invention, the micro fluidics kit is configured to provide the rapid indication with thirty seconds.
• According to another embodiment of the present invention, the micro fluidics kit is configured to provide the rapid indication with ten seconds.
• According to another embodiment of the present invention, the micro fluidics kit is configured to provide the rapid indication with one second.
• 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.
• There is thus provided according to an embodiment of the present invention, a microfluidics assay kit for performing a rapid assay of a biological entity, the kit comprising;
• • a) a disposable element comprising a reactant, the disposable element being adapted to receive a sample comprising the biological entity and for combining said reactant with said biological entity to form a reaction product; and
  • b) at least one reporter element adapted to provide a rapid indication of appearance of said reaction product thereby providing rapid assay of the biological entity.
• There is thus provided according to an embodiment of the present invention, a composition for evaluating a biological condition, the composition comprising;
• • a. a sample composition comprising at least one of;
    • i. a bodily specimen comprising a target moiety;
    • ii. a positive control moiety; and
    • iii. a negative control moiety;
  • b. a detection composition comprising at least one of;
    • i. at least one target antibody;
    • ii. at least one positive control identifying antibody; and
    • iii. at least one negative control identifying detection moiety or characteristic; and
  • c. at least one reference composition comprising at least one of;
    • i. a target signal reference composition; and
    • ii. a reference identifier composition.
• 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.
• 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.
• 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.
• 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.
• According to another embodiment of the present invention, the target moiety includes a CD64 surface antigen on neutrophils.
• Additionally, according to a further embodiment of the present invention, the positive control moiety includes monocytes and the negative control includes lymphocytes.
• Additionally, according to an embodiment of the present invention, the target moiety is CD64 on neutrophils, the positive control moiety includes CD64 expression on monocytes, and the negative control moiety includes lymphocytes without CD64 expression.
• 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.
• Yet further, according to an embodiment of the present invention, the at least one reference composition includes beads.
• Additionally, according to an embodiment of the present invention, the beads include polystyrene microbeads.
• 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.
• Furthermore, according to an embodiment of the present invention, the first fluorescent signal includes FITC and the second fluorescent signal includes Starfire Red fluor.
• 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.
• Furthermore, according to an embodiment of the present invention, the biomarker is a sepsis biomarker.
• Moreover, according to an embodiment of the present invention, the biomarker is CD64 or CD163.
• Additionally, according to an embodiment of the present invention, the sample is a blood sample.
• 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.
• Further, according to an embodiment of the present invention, the binding moiety is an antibody.
• According to an embodiment of the present invention, the software is capable of recognizing a specific lot of fluorescently-labeled particles.
• 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.
• 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.
• 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.
• Furthermore, according to an embodiment of the present invention, the binding moiety is anti-CD64 antibody and the first cell population includes polymorphonuclear leukocytes.
• Yet further, according to an embodiment of the present invention, the second cell population includes monocytes.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• Additionally, according to an embodiment of the present invention, the sepsis marker is CD64.
• Furthermore, according to an embodiment of the present invention, a second sepsis marker is CD163.
• 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.
• Further, according to an embodiment of the present invention, the sepsis marker is CD64 and the second sepsis marker is CD163.
• Additionally, according to an embodiment of the present invention, the binding moiety is an antibody.
• 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.
• 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.
• According to another embodiment of the present invention, the particles include the same fluorescent label as the fluorescently-labeled binding moiety.
• 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.
• 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.
• Notably, according to an embodiment of the present invention, the calibration is completed in less than 5 minutes.
• According to some embodiments, the particles are microbeads.
• Additionally, according to an embodiment of the present invention, the method is performed in less than 15 minutes.
• 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.
• 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
• 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.
• 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.
• In the drawings:
• FIG. 1 is a simplified schematic illustration showing an apparatus for detecting a biological condition, in accordance with an embodiment of the present invention;
• FIG. 2 is a simplified flow chart of a method for detecting a biological condition, in accordance with an embodiment of the present invention;
• FIG. 3 is a simplified schematic illustration showing a methodology for detecting a biological condition associated with a CD64 cell surface antigen, in accordance with an embodiment of the present invention;
• FIG. 4 is a simplified flow chart of a method for detecting a biological condition associated with a CD64 cell surface antigen, in accordance with an embodiment of the present invention;
• FIG. 5A is a graphical output of a fluorescent detection assay of a non-activated neutrophil signature associated with the method ofFIGS. 3-4, in accordance with an embodiment of the present invention;
• FIG. 5B is a graphical output of a fluorescent detection assay of an activated neutrophil signature, associated with the method ofFIGS. 3-4, in accordance with an embodiment of the present invention;
• FIG. 5C is a graphical output of a fluorescent detection assay of a monocyte signature, associated with the method ofFIGS. 3-4, in accordance with an embodiment of the present invention;FIG. 5D is a graphical output of a fluorescent detection assay of a reference bead signature, associated with the method ofFIGS. 3-4, in accordance with an embodiment of the present invention;
• FIG. 6 is a simplified flow chart of a method for differentiating between different particles, in accordance with an embodiment of the present invention;
• FIG. 7 is a graphical output of fluorescence from reference beads in eight wavebands, in accordance with an embodiment of the present invention;
• FIG. 8 is a graphical output of data fromFIG. 7 after a first mathematical manipulation, in accordance with an embodiment of the present invention;
• FIG. 9 is a graphical output of data fromFIG. 7 after a second mathematical manipulation, in accordance with an embodiment of the present invention;
• FIG. 10 is a graphical output of data fromFIG. 7 after a third mathematical manipulation, in accordance with an embodiment of the present invention; and
• FIG. 11 is a graphical output of an event locator, based on data fromFIG. 8-10, in accordance with an embodiment of the present invention.
• In all the figures similar reference numerals identify similar parts.
DETAILED DESCRIPTION OF THE INVENTION
• 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.
• 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.
• The microfluidic cartridges of the present invention may be any suitable cartridge as shown in the figures or any of the prior art cartridges described or cited herein, such as, but not limited to, those described in U.S. Pat. No. D669,191 S1, US20120266986 A1, EP1846159 A2, US2012275972, WO11094577A, US2007292941A and EP1263533 B1.
• Reference is now made toFIG. 1, which is a simplified schematic illustration showing anapparatus100 for detecting a biological condition, in accordance with an embodiment of the present invention.
• Apparatus100 is a kit comprising acartridge102 and a number of chemical/biochemical reactants termed herein, treatment compositions. The treatment compositions are adapted to react, at least in part, with biological specimen, such as a body specimen, to be introduced to the apparatus. The body specimen may be a bodily fluid such as, but not limited to, blood, serum, plasma, urine, saliva, cerebrospinal fluid (CSF), serous fluid, peritoneal fluid and synovial fluid. Additionally or alternatively, the body specimen may be a solid such as a hair, a tooth part, a bone part or a piece of cartilage.
• Apparatus100 comprises aspecimen receiving element118, adapted to transfer the specimen to asample composition chamber104. The sample composition chamber comprises on ormore transfer elements105, adapted to transfer the specimen from the sample composition chamber to one or more other locations in the cartridge. In the non-limiting example shown inFIG. 1,transfer element105 is a conduit in fluid connection with atreatment chamber112.
• Additionally, the cartridge comprises a number oftreatment composition chambers106,108,110, adapted to respectively house a corresponding number oftreatment compositions120,122,124. These treatment compositions may be liquid, solid or combinations thereof.Apparatus100 is typically sold commercially as a kit with the treatment compositions disposed therein. In some cases, theapparatus100 may be adapted for a one-off test and may be disposable. In other cases, the apparatus may be re-used. A re-usable apparatus may be adapted to receive additional external compositions (not shown) or may have a plurality of treatment compositions, wherein only a portion is used for each test.
• The apparatus may be constructed and configured such that the treatment composition comprises proteins attached to a surface, such as to beads. A plurality of beads or other structural elements with proteins attached to their surfaces can be made by any one or more of the following methodologies: —
• • simple attachment such as by adsorption via electrostatic or hydrophobic interactions with the surface, entrapment in immobilized polymers, etc.
  • non-covalent or physical attachment;
  • covalent bonding of the protein to the bead surface
  • biological recognition (e.g., biotin/streptavidin).
  • requires two steps: a first layer is formed by silane chemistry such that the surface presents a reactive group (e.g., epoxy, amino, thiol, etc.), and a second layer (e.g., the protein to be immobilized or a linker molecule) is covalently attached via the immobilized reactive groups.
  • covalent attachment to functionalized polymer coatings on the interior of the device or linkage to the free end of a self-assembled monolayer (SAM) on a gold surface.
• 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.
• Cartridge102 further comprises at least onetransfer element107,109,111 in fluid communication with each respective of treatment composition chamber, each transfer element also being in fluid communication withtreatment chamber112. These elements are typically micro fluidic channels and may be designed for mixing, such as being tortuous in shape.
• Various methodologies for transferring the contents of the treatment composition chambers and the sample composition chamber via the transfer elements to the treatment chamber may be employed, some of which are known in microfluidics technologies. These include air blowing, suction, vacuuming, mechanical transfer, pumping and the like.
• Cartridge102 further comprises at least onetransfer element113 in fluid communication withtreatment chamber112 and with anevaluation chamber114.
• Optionally,evaluation chamber114 is further in fluid communication with atransfer element115, adapted to remove the contents of the evaluation chamber for disposal outside the cartridge. Alternatively, the evaluation chamber may have no external disposal means.
• Table 1 shows some representative applications ofapparatus100 and methods of the present invention.

• TABLE 1
  Applications of the apparatus and methods of this invention.

  			Typical Prior	This
  		Relevant	Art Laboratory	invention
  		Figures in	Turnaround	Turnaround
  	Type of	this	time (TAT)-	time
  Application	Test	invention	see references	(TAT)	References
  Application #1 -	Surface	FIGS. 1-2	4hours	10 minutes	U.S. Pat. No. 8,116,984,
  CD64 Infection	Marker	and 3-5D			Davis, B H et al,
  & Sepsis					(2006)
  1 - Fetal	Plasma	FIGS. 1-2	4 hours	10 minutes	Dziegiel et al.
  Hemoglobin	Protein	and 6-8D			(2006)
  Test
  2 - Low Platelet	Surface	FIGS. 1-2	4 hours	10 minutes	Segal, H. C., et al.
  Count	Marker	and 3-5D			(2005):
  3 - Resolving	Surface	FIGS. 1-2	4 hours	10 minutes	Guerti, K., et al.
  BLAST Flag for	Marker	and 3-5D
  hematology Lab
  4 - CD34 Stem	Surface	FIGS. 1-2	4 hours	10 minutes	Sutherland et al.
  Cell	Marker	and 3-5D			(1996)
  Enumeration
  Assay
  5 - Platelets	Surface	FIGS. 1-2	4 hours	10 minutes	Graff et al. (2002)
  Activation	Marker	and 3-5D			Divers, S. G., et al.
  Assay CD62					(2003)
  6 - D-dimer	Plasma	FIGS. 1-2	4 hours	10 minutes	Stein et al. (2004)
  (Bead based	Protein	and 6-8D			Rylatt, D. B., et al.
  protein)					(1983):
  7-	Surface	FIGS. 1-2	4 hours	10 minutes	Hillier et al. (1988)
  Chorioamnioitis	Marker	and 3-5D
  CD64
  8 - CD20 Cell	Surface	FIGS. 1-2	4 hours	10 minutes	Rawstron et al.
  Quantitation	Marker	and 3-5D			(2001)
  (Therapy					Cheson et al.
  Monitoring					(1996)
  9 - CD52 Cell	Surface	FIGS. 1-2	4 hours	10 minutes	Rawstron et al.
  quantitation	Marker	and 3-5D			(2001)
  (Therapy
  Monitoring)
  10 - Circulating	Surface	FIGS. 1-2	4 hours	10 minutes	Cristofanilliet al.
  Tumor Cells	Marker	and 3-5D			(2004
  11 - Reticulated	Surface	FIGS. 1-2	4 hours	10 minutes	Matic et al. (1998)
  Platelet Assay	Marker	and 3-5D			Ault et al (1993)
  					Wang et al. (2002)
  12 - Bacteria			4 hours	10 minutes	Blajchman et al
  Detection in					(2005)
  platelet packs					McDonald et al.
  					(2005)
  13 - Platelet	Surface	FIGS. 1-2	4 hours	10 minutes	Michelson (1996)
  Associated	Marker	and 3-5D
  Antibodies
  14 - Residual	Surface	FIGS. 1-2	4 hours	10 minutes	Bodensteiner,
  Leukocyte	Marker	and 3-5D			(2003)
  Count in blood
  products
  15 - CD4 HIV	Surface	FIGS. 1-2	4 hours	10 minutes	Rodriguez (2005).
  AIDS	Marker	and 3-5D			Dieye et al. (2005)
  16 - Leukemia	Surface	FIGS. 1-2	4hours	10 minutes	Drexler et al (1986)
  Panels - Very	Marker	and 3-5D
  complex
  17 - Bladder	Surface	FIGS. 1-2	4hours	10 minutes	Ramakumar et al
  Cancer	Marker	and 3-5D			(1999)
  Screening in					Lotan et al. (2009)
  Urine - Urine
  sample
  18 - HLA DR	Surface	Figs. 1-2	4hours	10 minutes	Hershman et al.
  Sepsis and	Marker	and 3-5D			(2005)
  Immuno-					Perry et al (2003)
  suppression
  19 - RECAF	Plasma	FIGS. 1-2	4hours	10 minutes	Moro et al. (2005).
  Protein for	Protein	and 6-8D
  Canine and
  other Cancers
  20 - CytoImmun -			4hours	10 minutes	Hilfrich et al.
  Cervical					(2008)
  Screening
  21 -	Plasma	FIGS. 1-2	4hours	10 minutes	Assicot et al.
  Procalcitonin	Protein	and 6-8D			(1993)
  (Bead Based					Christ-Grain et al.
  Protein) +					(2004)
  Feasibility

• Reference is now made toFIG. 2, which is asimplified flow chart200 of a method for detecting a biological condition, in accordance with an embodiment of the present invention.
• 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.
• In a sample transferring step202, a sample, such as a bodily specimen is transferred fromoutside apparatus100 via receivingelement118 intosample composition chamber104 and then to thetreatment chamber112. 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.
• Thereafter,treatment composition120 is transferred viatransfer element107 to the treatment chamber in acomposition transfer step204. In some cases, there may be a treatment composition disposed in the treatment chamber.
• Depending on the nature of the treatment composition and sample/specimen type, there may be a requirement to mix or agitate the treatment chamber contents in anoptional mixing step206. This may be performed by using a small stirbar (not shown) disposed in the chamber. Additionally or alternatively, this may be effected by the fluid dynamics of kit. Additionally or alternatively, stirbars may be disposed in any of the other chambers in the apparatus.
• 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.
• According to some embodiments, the volume of thetreatment composition chambers106,108,110 (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.
• According to some embodiments, the volume of thetreatment compositions120,122,124 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.
• 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.
• The sequence of transfer of the various treatment compositions may be important to the reaction sequence and is typically predefined. Steps204-206 may be performed, for example ontreatment composition chamber106, thereafter ontreatment composition chamber108 and thereafter ontreatment composition chamber110. In some cases, some of these steps may be performed concurrently.
• In a checkingstep208, it is ascertained whether all the compositions required for the sample treatment have been transferred to the treatment chamber. If any compositions remain, then steps204-206 are performed on the subsequent treatment composition chamber(s). If no further treatment compositions require transfer, then the sample/specimen is transferred fromchamber104 into the treatment chamber.
• Thereafter, in a secondsample transfer step210, the sample is transferred from the sample composition chamber into the treatment chamber.
• According to some embodiments,step210 may be performed before steps204-208.
• If required, anoptional mixing step212 to the contents of the treatment chamber may be performed.
• In a transferringstep214, the contents of the treatment chamber are transferred to the evaluation chamber.
• Theevaluation chamber114 is configured and constructed for one or more evaluation steps216. 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.
• 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.
• The results of the evaluation step are then outputted in aresults outputting step218. 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 fromapparatus100.
• Reference is now made toFIG. 3, which is a simplified schematic illustration showing amethodology300 for detecting a biological condition associated with a CD64 cell surface antigen, in accordance with an embodiment of the present invention.
• According to some embodiments, the method is carried out in the apparatus shown inFIG. 1 and as described herein. A biological specimen, such as a blood sample, is aspirated viaspecimen receiving element118 to samplecomposition chamber104, and then totreatment chamber112. The sample is typically of a volume in the range of 10-200 μL.
• 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.
• Acocktail304 comprising antibodies to CD64 and antibodies to CD163 is introduced to the treatment chamber (see Davis et al. (2006)). Each antibody type is typically tagged by a specific fluorescent tag.
• The contents of the chamber are incubated and/or mixed as is required to bind the activated blood neutrophils with the CD64 tagged antibody (also called a marker) to form activated neutrophils withCD64 marker310, and/or monocyte with a CD64 tagged antibody and a CD163 taggedantibody312. Lymphocytes with nomarkers314 are present in the contents, as well asunaffected RBCs316.
• Thereafter, a lysis reagent ordiluent306 is introduced intotreatment chamber112. In the case of a lysis reagent, it is adapted to lyse red blood cells to form lysedred blood cells324. Additionally, reference/calibration beads308 are added to the treatment chamber. These are used to calibrate the outputs, as is explained with reference toFIGS. 5A-5D hereinbelow.
• CD64 (Cluster of Differentiation64) 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.
• CD163 (Cluster of Differentiation163) is a human protein encoded by the CD163 gene. It has also been shown to mark cells of monocyte/macrophage lineage.
• Reference is now made toFIG. 4, which is a simplified flow chart400 of a method for detecting a biological condition associated with a CD64 cell surface antigen, in accordance with an embodiment of the present invention.
• According to some embodiments, the method is carried out in the apparatus shown inFIG. 1 and as described herein. In afirst transferring step402, a biological specimen, such as a blood sample is aspirated viaspecimen receiving element118 to samplecomposition chamber104. The sample is typically of a volume in the range of 10-200 μL.
• 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.
• According to some embodiments, the volume of thetreatment composition chambers106,108,110 (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.
• According to some embodiments, the volume of thetreatment compositions120,122,124 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.
• 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.
• In anaddition step404, a cocktail of tagged antibodies to CD64 and to CD163 is added to thetreatment chamber112 and is incubated with the blood sample. In the incubation phase of this step, the antibodies bind activated neutrophils withCD64 marker310, and/or monocytes activated with a CD64 tagged antibody and a CD163 taggedantibody312.
• In a lysisreagent addition step406, the lysis reagent is added to the treatment chamber and thereby lyses at least some of the RBCs in the chamber.
• At any suitable time, typically followinglysis step406, reference beads are added to the contents of the treatment chamber in a referencebead adding step408.
• After a predefined period of time, ananalysis step410 is performed to analyze the fluorescent emission signatures from the contents. This is described in further detail with reference toFIGS. 5A-5D. According to some examples, theevaluation chamber114 is constructed and configured to allow cells to pass through areading zone130 such that each cell passing therethrough is analyzed individually. The assay sensitivity is around 86% and its specificity is around 87% (Hoffmann, 2011).
• The time required to complete anassay using apparatus100 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.
• Reference is now made toFIG. 5A, which is a graphical output of a fluorescent detection assay of a non-activatedneutrophil signature500 associated with the method ofFIGS. 3-4, in accordance with an embodiment of the present invention. The non-activated tagged neutrophils each emit asignal502 at wavelength W1 of an intensity I₁. The wavelengths shown inFIGS. 5A-5D represent a peak wavelength of waveband outputs detected, as are shown inFIGS. 7-11.
• FIG. 5B shows a graphical output of a fluorescent detection assay of an activatedneutrophil signature510, associated with the method ofFIGS. 3-4, in accordance with an embodiment of the present invention. Each activated tagged neutrophil emits an activatedneutrophil signature512 at wavelength W1 of an intensity I₂. Typically I₂is greater than I₁. In some cases the difference insignatures512 and510 may be detected by an image analysis, a fluorescent emission radiation count or by other qualitative or quantitative methods known in the art. The current example is not meant to be limiting.
• Turning toFIG. 5C, there can be seen a graphical output of a fluorescent detection assay of amonocyte signature520, associated with the method ofFIGS. 3-4, in accordance with an embodiment of the present invention. The monocyte signature comprises afirst signal522 at a first wavelength W1 of an intensity I₃and asecond signal524 at a second wavelength W2 of an intensity I₄.
• FIG. 5D shows a graphical output of a fluorescent detection assay of areference bead signature530, associated with the method ofFIGS. 3-4, in accordance with an embodiment of the present invention. The reference bead signature comprises afirst signal532 at a first wavelength W1 of an intensity I₁(similar or equal to non-activated tagged neutrophils' signal502) and asecond signal534 at a second wavelength W3 of an intensity I₅.
• This methodology enables the identification and quantification of activated neutrophils by intensity ofsignature512 of the CD64 tag. Monocytes are identified by thedouble signal signature522,524, acting as a positive control. Reference beads are identified by theunique signal534 at wavelength W3. The intensity ofsignal532 at wavelength W1 provides a reference level of CD64 for the comparison of intensity of 512 of the neutrophils.
• Lymphocytes with no markers330 (FIG. 3) act as a negative control and should provide no fluor signature, but may be detected by their scattering or other characteristics. Further details of some embodiment of this assay procedure are described in U.S. Pat. No. 8,116,984 and in Davis, B H et al., (2006).
• Reference is now made toFIG. 6, which is a simplified flow chart of amethod600 for differentiating between different particles, in accordance with an embodiment of the present invention.
• The input to the processing is a time series from each of the channels in the eightchannel photomultiplier array601. In addition, data frommultiple scatter channels609 is introduced. Each fluorescent time series and scatter time series may be processed individually employing respectivespectral crosscorrelation algorithm606 andscatter algorithm607 to smooth it and minimize noise. Two possible processing methods areboxcar averaging algorithm602 and matchedfiltering algorithm604. In addition, groups of individual channels may be correlated to yield a multiplespectral crosscorrelations606. One or more of these derived time series may be used to determine event locations.
• 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 meansquare error fit610. 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 itsdescription612. Based on the fluor composition for each event in the data store, it is possible to determine the type of particle. For example, aneutrophil616 is characterized by the single fluor attached to the CD64 antibody shown inFIG. 5 as W1. Thus events that are preponderantly characterized by the single fluor attached to the CD64 antibody are identified as neutrophils.
• Similarly,monocytes618 are characterized by fluors W1 and W2 so that an event with both of these fluor signatures is identified as a monocyte. Similarly, abead620 is characterized by an event that has fluors W1 and W3.Lymphocytes622 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 rejects626.
• 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.
• FIG. 7 is agraphical output700 of fluorescence from reference beads in eight wavebands, in accordance with an embodiment of the present invention. This figure shows the smoothed signals from the eight channel PMT array for two reference beads. The amplitude for each waveband is shown on the same graph. The corresponding wavelength range is shown for eachplot702,706,708,710,712,714,716,718 in the legend box. The two fluorophores signatures present in this plot are702,706 and708 for FITC, which is the fluorophore attached to the CD64 antibody and710,712 for Starfire Red, which is the fluorophore identifying the reference beads.
• Reference is now made toFIG. 8, which is agraphical output800 of data fromFIG. 7 after a first mathematical manipulation, in accordance with an embodiment of the present invention.FIG. 8 shows the cross correlation of wave bands one two and three corresponding towavelength 500 to 525, 525 to 550, and 552 to 575 nm. This cross correlation is computed by multiplying the boxcar smoothed time series corresponding to these wavelengths. This signal will have a high-value when an event containing the FITC fluorophore is present.
• FIG. 9 is agraphical output900 of data fromFIG. 7 after a second mathematical manipulation, in accordance with an embodiment of the present invention.FIG. 9 shows the cross correlation ofwave bands 3, 4 and 5 corresponding to wavelengths 550 to 575, 575 to 600, and 600 to 625 nm. This signal will have a high-value when an event containing the PE fluorophore is present.
• FIG. 10 is agraphical output1000 of data fromFIG. 7 after a third mathematical manipulation, in accordance with an embodiment of the present invention.FIG. 10 shows the cross correlation ofwave bands 7 and 8 corresponding to wavelengths 650 to 675, and 675 to 700 nm. This signal will have a high-value when an event containing the Starfire Red fluorophore is present.
• FIG. 11 is agraphical output1100 of an event locator, based on data fromFIG. 8-10, in accordance with an embodiment of the present invention.FIG. 11 shows the event locations determined from the cross correlations computed inFIGS. 8,9 and10. Thesolid fill area1102 corresponds to the region where any of thecross correlations802,902 and1002 exceeded a predefined threshold. Similarly, the solid fill area1104 corresponds to the region where any of thecross correlations804,904 and1004 exceeded a predefined threshold. This then completes the event location process.
ExampleApplication No. 1—CD64 Infection & Sepsis
• A cartridge102 (FIG. 1) is prepared for receiving a blood sample. The cartridge comprises a number oftreatment composition chambers106,108,110, adapted to respectively house a corresponding number oftreatment compositions120,122,124. 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 <0.1% sodium azide and 0.01% Tween 20).
• In a sample transferring step202 (FIG. 2), a 10 uL blood sample, is transferred fromoutside apparatus100 via receivingelement118 intosample composition chamber104 and then on totreatment chamber112 in a transferringstep214.
• An antibody composition (Reagent A)120 comprising CD64 antibodies is transferred viatransfer element107 to thetreatment chamber112 in acomposition transfer step204.
• These two steps combined with mixingstep206 take around fourminutes using cartridge102 of the present invention.
• A lysis buffer (Reagent B)122 is also added and mixed with the resultant mixed composition. This step and mixing all the compositions takes around threeminutes using cartridge102 of the present invention. Reference beads (Reagent C)308 are added to the treatment chamber.
• Theevaluation chamber114 is configured and constructed for one or more evaluation steps216.
• According to some embodiments, the cartridge is introduced into a system as described in International patent application publication no. WO2011/128893 to Kasdan et al., incorporated herein by reference. This system has software associated therewith for computing the CD64 and CD163 indices on leukocytes.
• The results of the evaluation step are then outputted in aresults outputting step218. According to this example, the time taken from the introduction of the small blood sample to obtaining an indication of sepsis is less than 15 minutes, typically around 10 minutes.
• From a user point of view, the following steps are performed:
• • 1) The user adds drop of blood to thecartridge102 and seals it. (10 μL are metered out by microfluidics).
  • 2) Blister A (106) is pressed, releasing 100 μL of Reagent A. Mixing in the cartridge is controlled by the cartridge handling unit (CHU), followed by a 4-minutes incubation.
  • 3) Blister B (108) is pressed, releasing ˜250 μL of Reagent B. Mixing in the cartridge is controlled by the CHU, followed by a 3-5-minutes incubation.
  • 4) Magnetic stirbar is activated, stirring the bead suspension (Reagent C).
  • 5) Blister C (110) is pressed, releasing 100 μL of Reagent C. Mixing in the cartridge is controlled by the CHU. According to one example, Reagent A is a mixture of murine monoclonal antibodies—diluted 1:5 in buffered saline (PBS+0.5% BSA); Reagent B is a Trillium Lyse solution (at working concentration); Reagent C is a suspension of 5.2 μm polystyrene beads labeled with Starfire Red and FITC, diluted 1:100 in PBS+0.01% Tween 20.
  • 6) The sample is read by the optoelectronics core, and collected to the reading below.
  • 7) Data is analyzed automatically and result is presented.
  • 8) The cartridge is disposed as biohazard.

• TABLE 2
  Comparison of Prior art methodology with the methodology of the present
  invention for detecting sepsis using CD64 and CD163 antibodies.
  LeukoDx device- present invention

  		Volume
  Step	Description	(uL)	Duration (min)	comments
  1	Mixing blood and	Blood- 10	4
  	antibodies	Abs- 50
  2	AddingRBC 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

• 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.
• According to some embodiments, the readout may comprise an optoelectronics core, which enables identification and detection of fluorescent signals.
• The CCD in the core, used for focusing, can also be used to read chemiluminescent signals. The readout to user may also indicate where the result falls relative to reference ranges.
• The contents of these publications are incorporated by reference herein where appropriate for teachings of additional or alternative details, features and/or technical background.
• It is to be understood that the invention is not limited in its application to the details set forth in the description contained herein or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Those skilled in the art will readily appreciate that various modifications and changes can be applied to the embodiments of the invention as hereinbefore described without departing from its scope, defined in and by the appended claims.
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Claims (43)

1-13. (canceled)

14. A method for determining a biological condition in a subject, the method comprising:

a. incubating a specimen from said subject in the kit of claim1 for a predetermined period of time; and

b. receiving an indication responsive to said at least one reporter element thereby providing the indication of the biological condition in said subject.

15. A method according toclaim 14, wherein the biological condition is selected from blood diseases such as leukemia, thrombocytopenia, immune system disorders, local infections, urinary tract disorders, autoimmune diseases and sepsis.

16. A method according toclaim 14, wherein said indication is quantitative.

17. A method according toclaim 14, wherein the sample is of a volume of less than 200 microliters (μL).

18. A method according toclaim 14, wherein the method is completed within twenty minutes.

19. A method according toclaim 18, wherein the method is completed within fifteen minutes.

20. A method for determining a biological condition in a mammalian subject, the method comprising:

a. incubating a specimen from the subject with at least one composition in a kit for a predetermined period of time to form at least one reaction product, when said subject has said biological condition; and

b. receiving an indication of said at least one reaction product responsive to at least one reporter element in said kit thereby providing the indication of the biological condition in said subject.

21. An automated method of determining the presence or absence of sepsis in a subject, comprising:

a. contacting a blood sample from the subject with a fluorescently-labeled binding moiety specific to a sepsis marker, wherein the volume of the blood sample is 50 μL or smaller;

b. detecting the presence, absence or level of the binding moiety in the sample, thereby determining the presence or absence of sepsis in the subject within twenty minutes.

22. The method ofclaim 21, wherein the sepsis marker is CD64.

23. The method ofclaim 21, wherein the sepsis marker is CD163.

24. The method ofclaim 21, further comprising contacting the blood sample with a second fluorescently-labeled binding moiety specific for a second sepsis marker.

25. The method ofclaim 24, wherein the sepsis marker is CD64 and the second sepsis marker is CD163.

26. The method ofclaim 21, wherein the binding moiety is an antibody.

27. The method ofclaim 21, wherein the detecting step is performed in a device capable of receiving the sample and capable of detecting the binding moiety.

28. The method ofclaim 24, further comprising the step of calibrating the device by detecting a population of the fluorescently-labeled particles.

29. The method ofclaim 28, wherein said particles comprise the same fluorescent label as the fluorescently-labeled binding moiety.

30. The method ofclaim 29, further comprising a second population of particles that comprise the same fluorescent label as the second fluorescently-labeled binding moiety.

31. The method ofclaim 28, further comprising performing an internal calibration after said detecting said fluorescently-labeled binding moiety.

32. The method ofclaim 31, wherein the calibration is completed in less than 5 minutes.

33. The method ofclaim 32, wherein the particles are microbeads.

34. The method ofclaim 21, wherein said method is performed in less than 15 minutes.

35. The method ofclaim 31, wherein the particles are microbeads.

36. The method ofclaim 21, further comprising 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.

37. A disposable kit for rapid evaluation of a biological condition in a subject, the kit comprising:

a static disposable cartridge adapted to receive a biological specimen from said subject, said cartridge including

at least one treatment composition chamber adapted to house at least one composition,

a treatment chamber comprising at least one of a vortexing element and tortuous mixing means adapted to provide a rapid reaction of said specimen, said at least one composition to form a reaction product, when said subject has said biological condition,

a detection chamber adapted to receive said reaction product to provide an indication of said reaction product indicative of said biological condition, and

a plurality of valveless microfluidic channels, each adapted to convey at least one of said at least one treatment composition, said reaction product, at least one detector moiety and said reporter element between any two of said chambers,

wherein said chambers, elements and channels are activated according to a predefined sequence.

38. A kit according toclaim 37, further comprising instructions for obtaining an indication of the biological condition by using the kit.

39. A kit according toclaim 37, wherein said disposable cartridge comprises at least one of the following elements:

a reservoir;

a pump;

a conduit;

a miniaturized flow cell;

a transport channel;

a reading channel;

a microfluidic element;

a compressed gas holding element;

a compressed gas releasing element;

a nozzle element; and

a mixing element.

40. A kit according toclaim 39, wherein said disposable microfluidics cartridge comprises at least two of the elements.

41. A kit according toclaim 40, wherein said disposable microfluidics cartridge comprises at least three of the elements.

42. A kit according toclaim 37, wherein said at least one composition disposed in said cartridge comprises at least one of:

at least one target antibody;

at least one positive control identifying antibody; and

at least one negative control identifying detection moiety.

43. A kit according toclaim 37, wherein said at least one composition disposed in said cartridge comprises at least one reference composition comprising at least one of:

a target signal reference composition; and

a reference identifier composition.

44. A kit according toclaim 37, wherein said at least one composition disposed in said cartridge comprises:

a positive control moiety; and

a negative control moiety.

45. A kit according toclaim 37, wherein said at least one composition comprises a sepsis biomarker.

46. A kit according toclaim 45, wherein said biomarker comprises at least one of CD64 and CD163.

47. A kit according toclaim 37, wherein said predefined sequence is adapted to occur within fifteen minutes.

48. A kit according toclaim 47, wherein said rapid evaluation is adapted to occur within fifteen minutes.

49. A kit according toclaim 37, wherein the biological condition is selected from blood diseases such as leukemia, thrombocytopenia, immune system disorders, local infections, urinary tract disorders, autoimmune diseases and sepsis.

50. A kit according toclaim 49, wherein the biological condition is sepsis.

51. A kit according toclaim 37, wherein said indication is a visual indication.

52. A kit according toclaim 50, wherein said visual indication is indicative of said biological condition.

53. A kit according toclaim 51, wherein said visual indication is provided within fifteen minutes.

54. A kit according toclaim 37, wherein the sample of said biological specimen is of a volume of less than 200 microliters (μL).

55. A kit according toclaim 37, wherein said at least one composition is of a volume of less than 200 microliters (μL).

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