Systems and Methods for Detecting a Biological Condition
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.
The present invention claims priority from US provisional patent application 61/737,854, to Kasdan et al, filed on Dec. 17, 2012, US provisional patent application 61/737,856, to Kasdan et al., filed on Dec. 17, 2012 and from US Patent Application no. 13/716,246 filed on December 17, 2012, incorporated herein by reference.
FIELD OF THE INVENTIONThe 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 INVENTIONThere 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 INVENTIONIt 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 self-contained system for performing an assay for determining a chemical state, the system including;
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- 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.
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.
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.
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.
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.
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.
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.
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.
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 mm2.
Notably, according to an embodiment of the present invention, the predetermined quantity is of a volume of 10 to 500 microliters.
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.
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.
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.
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.
Additionally, according to an embodiment of the present invention, the cartridge has a shelf-life of 6 to 24 months.
Importantly, according to an embodiment of the present invention, the cartridge is valveless.
Notably, according to an embodiment of the present invention, the assay is a flow cytometric assay.
Additionally, according to an embodiment of the present invention, the chemical state is a biochemical state.
Furthermore, according to an embodiment of the present invention, the biochemical state is indicative of a biological condition.
Additionally, according to an embodiment of the present invention, the sample is a biological sample.
In some cases, according to an embodiment of the present invention, the biological sample is a bodily sample.
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.
Additionally, according to an embodiment of the present invention, the cartridge is valveless.
Further, according to an embodiment of the present invention, the cartridge is a disposable microfluidics cartridge.
Yet further, according to an embodiment of the present invention, the sample is introduced to the cartridge via capillary action.
Additionally, according to an embodiment of the present invention, the cartridge includes at least one of the following elements;
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- 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.
Additionally, according to an embodiment of the present invention, the at least one reagent disposed in the cartridge includes at least one of;
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- a. at least one target antibody;
- b. at least one positive control identifying antibody; and
- c. at least one negative control identifying detection moiety.
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.
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.
Furthermore, according to an embodiment of the present invention, the at least one reagent disposed in the cartridge includes at least one sepsis biomarker.
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;
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- 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.
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.
Furthermore, according to an embodiment of the present invention, the indication is quantitative.
Moreover, according to an embodiment of the present invention, the method is completed within twenty minutes.
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;
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- 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.
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;
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- 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.
Furthermore, according to an embodiment of the present invention, wherein the sepsis marker is CD64.
Additionally, according to an embodiment of the present invention, the 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.
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;
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- 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.
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.
Additionally, according to an embodiment of the present invention the assay is a flow cytometric assay.
Further, according to an embodiment of the present invention, the chemical state is a biochemical state.
Further, according to an embodiment of the present invention, the biochemical state is indicative of a biological condition.
Furthermore, according to an embodiment of the present invention, the sample is a biological sample.
Additionally, according to an embodiment of the present invention, the biological sample is a bodily sample.
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.
Moreover, according to an embodiment of the present invention, the at least one reagent includes at least one of;
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- 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.
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.
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.
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.
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.
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.
Further, according to an embodiment of the present invention, the at least one reagent includes at least two reagents.
Moreover, according to an embodiment of the present invention, the at least two reagents include at least one of;
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- 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.
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.
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;
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- 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.
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;
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- 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;
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- 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 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;
- l) software adapted to activate said elements according to a specific sequence; and
- m) 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 microfluidics kit is configured to provide the rapid indication with thirty minutes.
According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with fifteen minutes.
According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with ten minutes.
According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with five minutes.
According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with one minute.
According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with thirty seconds.
According to another embodiment of the present invention, the microfluidics kit is configured to provide the rapid indication with ten seconds.
According to another embodiment of the present invention, the microfluidics 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;
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- 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.
- a. a sample composition comprising at least one of;
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.
- a. a sample composition comprising at least one of;
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;
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- 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.
- a. a sample comprising at least one of;
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;
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- 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.
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:
In all the figures similar reference numerals identify similar parts.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTSIn 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.
Reference is now made to
Shown in
The internal components of the reader assembly are shown in
Reference is now made to
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.
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.
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.
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.
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.
With respect to
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.
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
Reference is now made to
With respect to
Reference is now made to
Reference is now made to
A multiband dichroic mirror (not shown) similar, or identical to, mirror 552 of
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.
Turning to
Table 1 shows representative values for representative components for use in the present invention.
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.
Reference is now made to
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.
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.
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
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.
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
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.
Reference is now made to
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.
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
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 (
Reference is now made to
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.
In a body fluid introduction step 1004, part or all of the body fluid sample 6051 (
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 (
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 (
In a spectral emissions detection step 1010, a plurality of spectrally distinct signals is detected by multiple emission detector 454 (
Thereafter, in a data processing step 1012, the outputted data is processed by signal processor 6036 (
Turning to
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.
The instant invention includes software and algorithms for proper data analysis and conversion of raw fluorescence data into actual concentrations of relative biological markers.
In
In
In
In
In
In
In
In
An individual cell 1505 flows through a detection region 1510 in a microfluidic channel (seen as 1452,
A photomultiplier tube (PMT) array 1560 or avalanche diode array detects fluorescence at 8 different spatial locations corresponding to 8 spectral regions.
In a forming laser step 1602, a laser excitation beam shape is formed.
The excitation beam is reflected from a dichroic mirror 504 (
In a forward scatter measuring step 1606, the forward scatter from particles 1460 (
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.
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 (
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.
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.
Thereafter, in a dispersing step 1616, the dispersed pinhole or slit is dispersed and imaged onto a multi-element electrooptical detector (6034,
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.
Reference is now made to
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.
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.
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.
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.
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.
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.
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.
The method further comprises a spent sample disposal method 1858, for receiving a sample from the sample excitation/interaction processing.
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.
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.
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
Similarly, monocytes 618 are characterized by fluors W1 and W2 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 W1 and W3. 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.
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.
The middle frame 2730 shows separation of LY from NE based on Waveband6 (A610).
The bottom frame 2740 shows separation of beads from cells based on Waveband8 (Starfire Red).
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.
The algorithm in
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.
In a second step 1320, an analysis of a histogram of an SFR signature values as shown in
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.
In an analyzing PE histogram set step, 1344, the data is manipulated to find a group corresponding to monocytes.
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.
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.
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.
In an order by Diodel signature step 1382, dataset 1380 is analyzed according to a Diodel signature (see above). Dataset 1380 is then analyzed in an analyzing step 1384 to find a group of data having properties of neutrophils.
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.
Reference is now made to
In a first ordering signature step 1305 a first signature is used to order the dataset of waveband and signature values 1303.
In a second step 1321, an analysis of a histogram of a 1st signature values to find the group corresponding to 1st signature 1325, as exemplified in
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.
In an analyzing 2nd signature histogram set step, 1345, the data is manipulated to find a group corresponding to the 2nd signature.
In an extracting 2nd signature dataset step 1347, a 2nd signature dataset of waveband and signature values 1349 is extracted. A dataset of waveband and signature values with 1st and 2nd signatures groups removed 1361 is then further processed as follows. Set 1361 is organized according to its ith signature in an order according to ith signature ordering step 1363.
In an analyzing ith histogram to find a group corresponding to ith signature step 1365, set 1361 is analyzed to determine if any of the data have behavior corresponding to the ith signature.
In an ith signature extraction step 1367, an ith 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 1st 2nd and ith signature groups removed 1381.
In an order by Nth signature step 1383, dataset 1381 is analyzed according to an Nth 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.
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.
Referring to
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
As noted in
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
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
As described above, an upper group 1704 (
Within each group various parameters may be computed from the fields in the dataset. An example is shown in the following table.
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 MEDWaveband2 N 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.
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
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.
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's registers and/or memories into other data similarly represented as physical quantities within the computer's registers and/or memories or other information storage medium that may store instructions to perform operations and/or processes.
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.
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.
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.
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-RAY), DVD, or the like.
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.
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.
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.
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.
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.
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.
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's computer, partly on the user's computer, as a stand-alone software package, partly on the user'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'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).
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.
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.
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.
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.
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.
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.
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.
Table 2 shows some representative applications of apparatus 100 and methods 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.
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.
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 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.
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.
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.
The reading region 1450 (
-
- 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.
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.
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.
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.
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.
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 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.
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.
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 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.
EXAMPLEApplication No. 1-CD64 Infection & Sepsis
A cartridge 110 (
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
1. A flow cytometric assay method for determining a biological condition in a subject, the method comprising:
- a. incubating a sample from said subject in a self-contained stationary cartridge housing at least one composition and at least one reporter functionality, adapted to report a reaction of said at least one composition with said sample, with said at least one composition and with said at least one reporter functionality to report an assay of said reaction;
- b. optically detecting said reaction in a moving fluid;
- c. processing data outputted in said optically detecting step to determine said assay, comprising: i. processing a time series from each channel of an at least eight channel photomultiplier array or light receiving reader unit and scatter channels data; ii. characterizing at least one event in terms of a fluor composition thereof to output at least one derived time series; and iii. detecting at least one particle type from said at least one event associated with said at least one derived time series; and
- d. receiving an indication responsive to said assay, thereby providing an indication of the biological condition in said subject.
2. A method according to claim 1, wherein said at least one composition is wet.
3. A method according to claim 1, wherein said at least one composition is dry.
4. A method according to claim 1, wherein said at least one particle type is from said sample, said at least one particle type selected from the group consisting of: a neutrophil, a monocyte, a bead, a lymphocyte, a reject and combinations thereof.
5. A method according to claim 1, wherein said sample is in a form selected from the group consisting of: a solid, a powder, a crystal form, a liquid, a colloid, a suspension and combinations thereof.
6. A method according to claim 1, wherein said optically detecting step comprises:
- i. providing an excitation beam to said cartridge;
- ii. measuring a forward scatter measurement from particles in at least one of said sample and said at least composition in an optical reader;
- iii. passing a returned beam from said sample via a high-pass filter and a concave grating; and
- iv. detecting a plurality of at least eight spectrally distinct signals associated with said at least one reporter functionality produced by said concave grating in said at least one of photomultiplier array (PMT) and a light-receiving reader unit and scatter channels data.
7. A method according to claim 1, wherein said, wherein the biological condition is selected from blood diseases, leukemia, thrombocytopenia, immune system disorders, local infections, urinary tract disorders, autoimmune diseases and sepsis.
8. A method according to claim 1, wherein said optically detecting step comprises detecting a signal associated with at least one reporter functionality, said at least one reporter functionality adapted to report a reaction of said at least one composition with said sample, herein said at least one composition comprises: wherein said reagent bound to said solid support is selected from the group consisting of an immobilized enzyme, an immobilized substrate, a plasma protein bead, an antibody bead and an antigen bead.
- i. a cell surface marker;
- ii. a cell stain;
- iii. a reagent bound to a solid support;
- iv. a chemical indicator; and
- v. a biological cell indicator;
9. A method according to claim 8, wherein said 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.
10. A method according to claim 8, wherein said at least one composition comprises at least two compositions.
11. A method according to claim 10, wherein said at least two compositions comprise 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.
12. A method according to claim 8, wherein said at least one composition comprises:
- a) 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
- b) at least one reference composition comprising at least one of; i. a target signal reference composition; and ii. a reference identifier composition.
13. A method according to claim 8, wherein said at least one composition comprises:
- a) an antibody composition comprising at least one of; i. at least one target antibody; ii. at least one positive control identifying antibody; and iii. at least one negative control identifying antibody or characteristic; and
- b) at least one reference composition comprising at least one of; iv. a target signal reference composition; and v. a reference identifier composition.
14. A method according to claim 8, wherein said sample comprises at least one of;
- i. a bodily specimen comprising a target moiety;
- ii. a positive control moiety; and
- iii. a negative control moiety.
15. A method according to claim 8, wherein the at least one composition further comprises at least one conditioning moiety comprising;
- a. at least one lysis reagent; and
- b. at least one diluent.
16. A method according to claim 6, wherein said providing step further comprises passing said excitation beam via a dichroic mirror through an objective onto a reading channel in said cartridge.
17. A method according to claim 16, further comprising, collecting particle fluorescent emission through said objective, further passing said particle fluorescent emission through said dichroic mirror and reflecting said fluorescent emission from a beamsplitter into a detection path.
18. A method according to claim 1, wherein said receiving an indication step further comprises outputting bandwidth leveled and smoothed arrays.
19. A method according to claim 18, wherein said receiving an indication step further comprises outputting a three-dimensional graph showing optical output over time from said optically detecting step.
20. A method according to claim 18, wherein said receiving an indication step further comprises providing a graphical display showing a cluster analysis of wavebands from at least some of said eight channels.
21. A method according to claim 20, further comprising applying an algorithm to said cluster analysis to provide a detection of at least one signature associated with said biological condition.
Type: Application
Filed: Jul 11, 2017
Publication Date: Dec 28, 2017
Inventors: Harvey Lee Kasdan (Jerusalem), Julien Meissonnier (Jerusalem), Yoav Zuta (Jerusalem), Micha Rosen (Jerusalem), Yael Himmel (Jerusalem), Yehoshua Broder (Jerusalem), Bruce Davis (Jerusalem), Bruce Goldman (Jerusalem), Boaz Giron (Jerusalem), Zion Botesazan (Jerusalem), Ellezer Blasberg (Jerusalem), Ilan Semmel (Jerusalem), Jacques Aschkenasy (Jerusalem)
Application Number: 15/646,111
