Methods and systems for imaging of labeled cells

Abstract

Disclosed are methods and systems for analyzing fluid samples comprising a plurality of cells. In particular, disclosed are methods for analyzing a fluid sample by labeling a subset of cells in the fluid sample with one or more nanoparticle conjugates, imaging individual cells in the fluid sample via detection of one or more inducible colors associated with one or more nanoparticle conjugates. The images obtained may be analyzed to identify labeled cells. Also disclosed are methods of preparing a fluid sample for such analysis and systems for implementing such analysis.

Description

METHODS AND SYSTEMS FOR IMAGING OF LABELED CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Patent Application Serial No.63 / 650,737, filed on May 22, 2024.FIELD

[0002] Disclosed are compositions, methods, and systems for analyzing particles in a fluid sample. In particular, disclosed are compositions, methods, and systems for the detection, quantification, and / or phenotyping of cells in a fluid sample using nanoparticles having specific binding to a target protein, such as a cell-surface antigen, on a cell in a fluid sample, wherein the nanoparticles can be detected via imaging. In aspects, the imaging used is brightfield imaging. The disclosed methods may be used to detect, quantify, and / or phenotype cells in a sample using a wholly or partly automated device.BACKGROUND

[0003] Blood cell analysis is one of the most commonly performed medical tests for providing an overview of a patient’s health status. A blood sample can be drawn from a patient’s body and stored in a test tube containing an anticoagulant to prevent clotting. A whole blood sample normally comprises three major classes of blood cells including red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). Each class can be further divided into subclasses of members. For example, five major types or subclasses of white blood cells (WBCs) have different morphologies and functions. White blood cells may include neutrophils, lymphocytes, monocytes, eosinophils, and basophils. There are also subclasses of the red blood cell types. Red blood cell subclasses may include reticulocytes and nucleated red blood cells. Lastly, blast cells, immature blood cells typically found in bone marrow which are precursors to various types of mature blood cells, can indicate abnormalities or malignancies when present in excessive amounts in the bloodstream. Thus, detection and analysis of cells in a sample can provide valuable diagnostic information.

[0004] A blood cell count estimating the concentration of RBCs, WBCs, platelets, or blast cells can be done manually or using an automatic analyzer. When blood cell counts are done manually, a drop of blood is applied to a microscope slide as a thin smear. Traditionally, manualexamination of a dried, stained smear of blood on a microscope slide has been used to determine the number or relative amounts of the five types of white blood cells. Histological dyes and stains have been used to stain cells or cellular structures. For example, Wright’s stain is a histologic stain that has been used to stain blood smears for examination under a light microscope.

[0005] Cell analysis may also be performed using flow imaging. Flow imaging is a powerful technique for cell analysis that combines certain characteristics of flow cytometry with high-resolution imaging, in which cells are suspended in a fluid medium and injected into a flow cell within an instrument. As cells pass through a flow cell, they are illuminated with a light source and images are obtained. Flow imaging allows for quantification, visualization and identification of cells in a heterogeneous population. Cell populations and morphological characteristics may be evaluated using this technique.

[0006] However, within the established techniques of counting and analyzing cells, there are limitations. For instance, certain types of cells (e.g., blast cells) can be difficult to detect and enumerate. With respect to the use of flow imaging to detect, quantify, and / or phenotype cells, such methods may not permit differentiation among different subsets (subtypes) of similar cells based on an image. For instance, it may be difficult to distinguish lymphocyte cell types (e.g. T and B cells) which may visually look similar to each other (e.g., where image analysis is used) or have similar measurement characteristics (e.g., where impedance or light scattering is used). Because accurately identifying cell types is important for many reasons including diagnosis, tracking disease progression, monitoring treatment response, and advancing research and development, the need for improved methods of visualizing and identifying cell types is desirable. The instant disclosure seeks to address one or more of the aforementioned needs in the art.BRIEF SUMMARY

[0007] Disclosed are methods for analyzing a cell type in a fluid sample. In aspects, the method comprises incubating a fluid sample with a nanoparticle conjugate to form a subset of labeled cells; detecting a color of a nanoparticle conjugated to a binding agent (referred to herein as a “nanoparticle conjugate”) on the subset of labeled cells; and characterizing the labeled cell based on the presence or absence of the nanoparticle conjugate. The disclosed methods may further comprise flowing a sheath fluid along a flowpath of a flowcell; injecting the fluid sample into theflowpath of the sheath fluid within the flowcell; imaging the labeled cell along an imaging path to produce a plurality of images, the imaging comprising detection of a color of a nanoparticle bound to a labeled cell; and analyzing each of the plurality of images to identify labeled cells. Multiplex and single plex methods for the identifying cell types are disclosed.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] While the specification concludes with claims which particularly point out and distinctly claim the invention, it is believed the present invention will be better understood from the following description of certain examples taken in conjunction with the accompanying drawings, in which like reference numerals identify the same elements and in which:

[0009] FIG. l isa schematic illustration, partly in section and not to scale, showing operational aspects of an exemplary flow cell which may be used in an analyzer configured to capture and analyze images.

[0010] FIG. 2 is an image of cells contacted with nanoparticles. Red imaging indicates nanoparticles bound to the cell. Gold images indicate cells that do not have nanoparticles bound to the cells surface.

[0011] FIG. 3 depicts an example of the results of a single plex assay in which a metal nanoparticle (AuNP) conjugated to an alpha-CD14 antibody is contacted with a population of cells (top, left and right). The resulting cell is imaged via a black and white camera. The image shows the nanoparticles coalesced around a target cell. Control conditions without conjugated antibody are shown (bottom, left and right).

[0012] FIG. 4. depicts an exemplary color scheme for a plurality of nanoparticles and detectable wavelengths for each.

[0013] The drawings are not intended to be limiting in any way, and it is contemplated that various embodiments of the invention may be carried out in a variety of other ways, including those not necessarily depicted in the drawings. The accompanying drawings incorporated in and forming a part of the specification illustrate several aspects of the present invention, and together with the description serve to explain the principles of the invention; it being understood, however, that this invention is not limited to the precise arrangements shown.DETAILED DESCRIPTION

[0014] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. The methods may comprise, consist of, or consist essentially of the elements of the compositions and / or methods as described herein, as well as any additional or optional element described herein or otherwise useful in detecting, imaging, phenotyping, and / or otherwise assaying cell types in a fluid sample.

[0015] All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

[0016] The headings provided herein are not limitations of the various aspects or embodiments of the invention. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual (Table of Contents available at www.cshlpress.com / pdf / sample / 2013 / MC 4 / MC 4FM.pdf).

[0018] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of + / - 15%, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0019] As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, referenceto “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0020] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0021] The term “antibody” is well understood by those in the field. The term refers to a protein consisting of one or more polypeptides that specifically binds an antigen. One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions. These terms also include chimeric antibodies, humanized antibodies, single-chain antibodies, bi-specific hybrid antibodies. The term “specific binding” refers to the ability of a binding agent to preferentially bind to a particular analyte that is present in a homogeneous mixture of different analytes. In certain aspects, a specific binding interaction will have a limit of detection (LOD) of about 1 in 10,000. In aspects, the LOD is about 1 in 100,000 to about 1 in IM.

[0022] As used herein, the terms “molecule of interest,” “target molecule,” “target,” “target antigen,” “biomarker,” and “analyte of interest” are used interchangeably, each referring to a molecule for which the presence, location and / or concentration is to be determined. Examples of molecules of interest include proteins which are expressed on a cell surface.Imaging Methods

[0023] As described above, imaging techniques for assaying cells in a sample, particularly a blood sample, are known. Currently available techniques, however, do not always allow for positive identification of specific cells often required for diagnosis such as B-cells, T-cells, and blasts. For example, it can be difficult to obtain information via imaging alone because cells may appear similar in morphology (e.g. size and / or shape) despite being functionally different cell types. Further, there is a need in the art for improved disease detection methods, for example for the detection of disease states such as B-cell acute lymphoblastic leukemia, chronic lyphocytic leukemia, and diffuse large B-cell lymphoma. The instant disclosure provides compositions, methods, and systems for detection, quantification, and / or phenotyping of a cell, which may be performed using static or flow imaging techniques, and which allows for improved identification of cell types in a fluid sample. In further aspects, disclosed are methods by which a disease state can be diagnosed in an individual from which the cell sample is obtained, based on detection of nanoparticles bound to a target cell. In aspects, the disclosed methods employ brightfield imaging (also known as transmitted light microscopy or compound light microscopy) to illuminate nanoparticles bound to the cells in a sample, which is used to create an image of the nanoparticle-labeled cells for detection, quantification, and / or phenotyping of one or more target cells.

[0024] In aspects, the disclosed methods utilize a static imaging system such as a slide-based imaging system to detect, quantify, and / or phenotype a cell or cell population in a sample using a nanoparticle. In aspects, the disclosed methods utilize a static imaging system such as a slide-based imaging system to detect, quantify, and / or phenotype a cell or cell population in a sample using a nanoparticle for purposes of identifying a disease state in the individual from which the sample was obtained. The sample may be a fluid sample, such as a blood or serum sample or other biological sample suspected of having one or more cell types of interest. In aspects, the methods employ obtaining images of a nanoparticle-labeled cell on a solid substrate, such as a glass slide or a microfluidic chip, upon which the cells to be analyzed are fixed in place. Images of the cells may be obtained using transmitted light (brightfield microscopy) and high-resolution imaging under controlled conditions, allowing for visualization of cell type, cell number, cell morphology and structure, and / or cell phenotype representing a disease state in the individual. Analysis of theimages obtained may be performed automatically, i.e., using a computing device, or may be performed without a computer.

[0025] In aspects, the disclosed methods utilize a non-static system. For example, in aspects, the non-static system is a flow imaging system. Flow imaging methods typically comprise flowing a sheath fluid along a flowpath of a flowcell, injecting the fluid sample into the flowpath of the sheath fluid, and imaging the cells in the fluid sample along an imaging path to produce a plurality of images. In aspects, the fluid sample is injected into the interior of the flowcell at a point where the flowpath of the sheath fluid has been established, resulting in a stable and symmetric laminar flow of the sheath fluid. The fluid sample and the sheath fluid may be supplied, for example by precision metering pumps, along the flowpath, which narrows substantially. The narrowing of the flowpath can contribute to a geometric focusing of the fluid sample. The fluid sample is carried along with the sheath fluid and passes in front of, or otherwise along the imaging path, of an imaging device, which collects a plurality of images. In aspects, the fluid sample passes a light source and brightfield images are digitally captured. The fluid sample and sheath fluid continue to flow to a discharge.

[0026] FIG. 1 schematically shows an exemplary flow cell 22 which may be used in an analyzer for conveying a sample fluid through a viewing zone 23 of a high optical resolution imaging device 24 (e.g., a camera) in a configuration for imaging nanoparticle conjugates in a sample flow stream 32 using digital image processing. Flow cell 22 is coupled to a source of sample fluid 25 which may have been subjected to processing, such as contact with a nanoparticle conjugates and heating and / or incubation to aid in the conjugation process. Flow cell 22 is also coupled to one or more sources of a nanoparticle conjugate (e g., part of, or mixed with a sample fluid 25) and / or intracellular organelle alignment liquid (PIOAL)Zsheath fluid 27, such as a clear glycerol solution having a viscosity that is greater than the viscosity of the sample fluid, an example of which is disclosed in U.S. Pat. Nos. 9,316,635 and 10,451,612, the disclosures of which are hereby incorporated by reference in their entirety.

[0027] In aspects, the sample fluid is injected through a flattened opening at a distal end 28 of a sample feed tube 29, and into the interior of the flow cell 22 at a point where the PIO AL flow has been substantially established resulting in a stable and symmetric laminar flow of the PIO ALabove and below (or on opposing sides of) the ribbon-shaped sample stream. The sample and PIOAL streams may be supplied by precision metering pumps that move the PIOAL with the injected sample fluid along a flow path that narrows substantially. The PIOAL envelopes and compresses the sample fluid in the zone 21 where the flow path narrows. Hence, the decrease in flow path thickness at zone 21 can contribute to a geometric focusing of the sample flow stream 32. The sample flow stream 32 is enveloped and carried along with the PIOAL downstream of the narrowing zone 21, passing in front of, or otherwise through the viewing zone 23 of, the high optical resolution imaging device 24 where images are collected, for example, using a CCD 48. Processor 18 can receive, as input, pixel data from CCD 48. The sample fluid ribbon flows together with the PIOAL to a discharge 33. In counting techniques based on imaging, pixel data images of a prepared sample that may be passing through a viewing area are captured using a microscopy objective lens coupled to a digital camera. The pixel image data can be analyzed using data processing techniques and displayed on a monitor.

[0028] As shown here, the narrowing zone 21 can have a proximal flow path portion 21a having a proximal thickness PT and a distal flow path portion 21b having a distal thickness DT, such that distal thickness DT is less than proximal thickness PT. The sample fluid can therefore be injected through the distal end 28 of sample tube 29 at a location that is distal to the proximal portion 21a and proximal to the distal portion 21b. Hence, the sample fluid can enter the PIOAL envelope as the PIOAL stream is compressed by the zone 21, wherein the sample fluid injection tube has a distal exit port through which sample fluid is injected into flowing sheath fluid, the distal exit port bounded by the decrease in flow path size of the flow cell.

[0029] The digital high optical resolution imaging device 24 with objective lens 46 is directed along an optical axis that intersects the ribbon-shaped sample flow stream 32. The relative distance between the objective 46 and the flow cell 33 is variable by operation of a motor drive 54, for resolving and collecting a focused digitized image on a photosensor array. Additional information regarding the construction and operation of an exemplary flow cell such as shown in FIG. 1 is provided in U.S. Patent 9,322,752, entitled “Flow cell Systems and Methods for Particle Analysis in Blood Samples,” fded on March 17, 2014, the disclosure of which is hereby incorporated by reference in its entirety.

[0030] In operation, an analyzer such as an analyzer incorporating a flow cell-based imaging system as illustrated in FIG. 1 may analyze images of cells in a sample. Initially, cells from a sample (e.g., a blood sample or other type of biological sample) may be flowed 201 through a flow cell (e.g., the flow cell 22 depicted in FIG. 1). As the cells are being flowed 201 through the flow cell, a plurality of cell images may be captured 202 (e.g., using a camera 24 as described in the context of FIG. 1), in which each cell image may depict a cell as it is flowing through the flow cell. The cell images may then be processed to determine 203 a set of cell parameter values for each image. This processing may include, for example, separating the foreground from the background of each image (e.g., using thresholding, in which images are converted a format such as hue, saturation and lightness and the lightness values for those images are compared to a predetermined threshold). In this type of embodiment, after separating the foreground and background, the cell parameter values for an image may be determined 203 by analyzing the foreground portion of that image.

[0031] The imaging device collects a plurality of images, known as patches. In aspects, a patch includes an image of a single cell. The plurality of images, taken as a whole, may be used to provide individual images of each cell and / or the conjugates in the fluid sample as described herein. In some scenarios, for example when the fluid sample has a high concentration of cells, a patch may include an image of multiple cells.

[0032] The plurality of images, or patches, produced by the imaging device may be analyzed to identify cell type or to identify a cell phenotype that is representative of a disease state. In aspects, this analysis may be done manually, i.e., by visual inspection of each image. Patches including nanoparticle-labeled cells can be readily differentiated from patches that include cells not labeled with nanoparticles. Visual analysis of the patches therefore allows for the determination of both the total number of cells and / or particles in the fluid sample as well as the number of labeled cells, i.e., the number of cells in the selected subset.

[0033] Analysis of the images may be performed automatically, i.e., using a computing device. For example, a computer may implement an artificial intelligence protocol to analyze patches and discriminate labeled cells from unlabeled cells. Alternatively, a computer may utilize a pixel-based image analysis protocol (e.g., based on hue, saturation, and / or brightness pixel data) for thisanalysis, or a combination of artificial intelligence and pixel analysis. Tn these aspects, the computing device would be able to determine the total number of cells and / or particles in the fluid sample as well as the number of labeled cells.

[0034] After having been imaged, the fluid sample and the sheath fluid may continue to flow to a discharge. In aspects, the fluid sample may be subject to further treatment. The labeled cells may be physically isolated by, for example, utilizing magnetic particles and then subjecting the fluid sample to a magnetic column. The isolated cells may then be washed to remove the particles and subject to further analysis.

[0035] In aspects, disclosed are systems for imaging a plurality of cells in a biological sample or other biological sample obtained from an individual which contain cells to be analyzed. In particular, disclosed are systems for employing the analytical methods disclosed herein. The system may comprise a flowcell having a flowpath and a sample fluid injection tube; a sheath fluid input in fluid communication with the flowpath of the flowcell so as to transmit a flow of the sheath fluid along the flowpath of the flowcell; a biological sample input in fluid communication with the injection tube of the flowcell so as to inject a flow of the biological sample into the flowing sheath fluid within the flowcell such that the injected cells align transverse to the orientation of an imaging path; and an imager in communication with the imaging path, so at to produce multiple image patches of the plurality of cells along the imaging path at an imaging site.

[0036] Some aspects of the system comprise a flowcell for conveying a biological sample through a viewing zone of a high optical resolution imaging device in a configuration for imaging microscopic particles in a sample flow stream using digital image processing. In these aspects, the flowcell is coupled to a source of the biological sample, which may have been subjected to processing, such as the above-described method of labeling with nanoparticle conjugates. In these aspects, the flowcell is also coupled to one or more sources of sheath fluid.

[0037] In aspects, the biological sample is injected through a flattened opening at a distal end of the injection tube and into the interior of the flowcell at a point where the flow of the sheath fluid has been substantially established. This allows for a stable and symmetric laminar flow of the sheath fluid above and below (or on opposing sides of) the biological sample stream. The biological sample and the sheath fluid streams may be supplied by precision metering pumps thatmove the sheath fluid along with the injected biological sample along a flowpath that narrows substantially. The sheath fluid envelopes and compresses the sample fluid in the region where the flowpath narrows. Hence, the decrease in flowpath thickness in this region can contribute to a geometric focusing of the biological sample stream. The sample is enveloped and carried along with the sheath fluid downstream of the narrowing region, along the imaging path to pass into, or otherwise through, the imaging site of the high optical resolution imaging device where images are collected. The biological sample then flows together with the sheath fluid to a discharge.Nanoparticles and Nanoparticle Compositions

[0038] The methods and systems disclosed utilize nanoparticles and compositions comprising nanoparticles to analyze a cell in a fluid sample. Such methods may be carried out for identifying a cell type, or a cell phenotype that is representative of a disease state. In general, the disclosed nanoparticles may be conjugated to a binding agent, e.g., an antibody or fragment thereof which has binding specificity for a target antigen (such as a cell surface molecule or protein) on a cell, to form a “nanoparticle conjugate.” In general, the disclosed systems and methods employ a composition comprising at least one nanoparticle conjugate comprising a binding agent specific for a target cell antigen. In aspects, the composition may comprise more than one nanoparticle conjugate for detection of more than one target cell antigen (referred to interchangeably as “target cell protein”). The more than one target cell antigen may be on the same cell or on different cells. In aspects, the composition is a composition for the detection of more than one target antigen, and comprises at least two nanoparticle conjugates, each nanoparticle being conjugated to a different binding agent. In aspects, the composition comprises at least three nanoparticle conjugates, each nanoparticle being conjugated to a different binding agent. In aspects, the composition comprises at least four nanoparticle conjugates, each nanoparticle being conjugated to a different binding agent. In aspects, the composition comprises at least five nanoparticle conjugates, each nanoparticle being conjugated to a different binding agent. In aspects, the composition comprises at least six nanoparticle conjugates conjugated to a binding agent, each nanoparticle being conjugated to a different binding agent. In aspects, the composition comprises at least seven nanoparticle conjugates, each nanoparticle being conjugated to a different binding agent. In aspects, the composition comprises at least eight nanoparticle conjugates, each nanoparticle being conjugated to a different binding agent. In aspects, the composition comprises at least ninenanoparticle conjugates, each nanoparticle being conjugated to a different binding agent. In aspects, the composition comprises ten or more nanoparticle conjugates, each nanoparticle being conjugated to a different binding agent. In general, each nanoparticle associated with a particular binding agent will be visually distinct from another nanoparticle associated with a different binding agent. Typically, the nanoparticles are distinguishable based on color. In general, using the nanoparticles as disclosed herein, the nanoparticles are detectable after a sufficient number of nanoparticles are bound to the cell surface.

[0039] The compositions comprising nanoparticle conjugates may comprise any suitable carrier sufficient to maintain the integrity of the binding agent and nanoparticle. In one aspect, the nanoparticle conjugate is provided in a composition that is isotonic to the fluid sample being assayed. In one aspect, the nanoparticle conjugate is provided in a composition that is at physiological pH.

[0040] The size and shape of nanoparticles used in the disclosed methods may vary. In one aspect, the nanoparticle has a diameter of between about 1 and about 1000 nm. In one aspect, the nanoparticle has a diameter of between about 1 to about 1000 nm. In one aspect, the nanoparticle has a diameter of about 10 to about 750 nm. In one aspect, the nanoparticle has a diameter of about 15 to about 500 nm. In one aspect, the nanoparticle has a diameter of about 20 to about 250 nm. In one aspect, the nanoparticle has a diameter of about 25 to about 100 nm. In one aspect, the nanoparticle has a diameter of about 1 to about 100 nm.

[0041] The nanoparticles used with the disclosed systems and methods may have a range of unique properties including surface chemistry, size, shape-dependent electronic, and optical properties. A variety of nanoparticle types are known in the art. For example, nanoparticles known in the art include semiconductor nanoparticles, metal nanoparticles, and polymer / biomacromolecular nanoparticles. The nanoparticles contemplated herein may be of any material suitable for carrying out the disclosed methods and may comprise one or a combination of nanoparticles as described herein. Nonlimiting examples of each are described below.

[0042] Metal Nanoparticles

[0043] In aspects, a metal nanoparticle can be used with the disclosed methods, such that upon aggregation of the nanoparticle at the cell surface following binding to the cell, a detectable color is observed. Suitable metal nanoparticles include, for example, nanoparticles comprising noble metals, such as gold, silver, cobalt, or nickel. In certain aspects, the nanoparticle is a gold nanoparticle. Nanoparticles have an observable color, such as red, when examined in bright field microscopy due to the nanoparticle’s ability to scatter and absorb light at specific wavelengths, where the size, shape, and composition of the nanoparticles in conjunction with the environment associated with the brightfield observation influence the wavelength of light absorbed and scattered - resulting in different observable colors. When a large plurality of nanoparticles are aggregated or concentrated together, the observable color is more noticeable.

[0044] In aspects, the size of the nanoparticle can affect the emission of color of the nanoparticle. A range of nanoparticle sizes for use with the disclosed methods are contemplated. For example, in aspects, an exemplary nanoparticle is a gold nanoparticle having a size of from about 10 to about 75 nm. In further aspects, the nanoparticle is a gold nanoparticle having a size of from about 25 to about 45 nm. In further aspects, the nanoparticle is a gold nanoparticle having a size of from about 30 to about 40 nm. In further aspects, the nanoparticle is a gold nanoparticle having a size of about 40 nm (with about 15% size standard deviation (“SD”). In yet further aspects, the nanoparticle is a metal nanoparticle having a diameter of about 60 nm (about 15% size SD).

[0045] In aspects, the shape of the nanoparticle, in addition to size, can affect the emission of the color of the nanoparticle. For example, the nanoparticle may have a spherical, elongated, or roddike shape. More particularly, in aspects, the nanoparticle is a spherical gold nanoparticle having a mean diameter of about 40 to about 60 nm and which has an observable pink / red color in a brightfield environment. In aspects, the nanoparticle is a spherical silver nanoparticle with a mean diameter of about 40 to about 60 nm, and which has an observable yellow color in brightfield. In aspects, the nanoparticle is a gold nanorod having a length of about 50 to about 200 nm, and which has a tunable color, which is typically observed as blue in brightfield conditions but which can be tuned or visualized to brown depending on the length and width customization of the nanoparticle. In a yet further aspect, the nanoparticle is a gold nanorod that is blue with a length of about 57 nm and width of about 25 nm. Reference is made to FIG. 4, which depicts threedifferent nanoparticles which can be detected as three different colors under brightfield conditions: a first nanoparticle is a spherical shaped metal nanoparticle that emits a detectable yellow color; a second nanoparticle is an elongated metal nanoparticle that emits a detectable red color, and a third nanoparticle is a rod shaped metal nanoparticle that emits a detectable blue color. In one example, the first nanoparticle (AgNP) is a silver nanoparticle and has a detectable yellow color because the nanoparticle has a high absorption of lower wavelength light (shown by the spike in the blue region of the wavelength spectrum), resulting in a detectable yellow color associated with blue’s complementary color. The second nanoparticle (AuNP) is a gold nanoparticle configured such that the nanoparticle has a high absorption of a median wavelength light (shown by the spike in the green region of the wavelength spectrum), resulting in a detectable red color associated with green’s complementary color. The third nanoparticle (AuNR) is a gold nanoparticle configured such that nanoparticle has a high absorption of median and especially high wavelength light (shown by the smaller spike in the green region of the wavelength spectrum and the larger spike in the red region of the wavelength spectrum), resulting in a detectable particle associated with a blue complementary color.

[0046] It will be appreciated that aggregation or concentration at the cell surface enhances the ability to detect the nanoparticle color. The particles have an observable wavelength when bound to a cell in aggregate (for example, of about 480 nm) despite being about 20-40 nm because the observed color is based on the material and the shape of the nanoparticle, in combination with the aggregation of nanoparticles after binding to a cell. In aspects, using the disclosed methods, the nanoparticles bind to a target cell via the cell surface and concentrate at the surface of the cell. In aspects, binding to a cell surface includes specific binding to a target antigen, such as a protein. In aspects, when bound to a cell the nanoparticles concentrate at the cell membrane and create a detectable color “halo” around the cell. A black and white image of this aggregation, showing the density of nanoparticles on a cell surface, is shown in FIG. 3. It will be appreciated that the disclosed nanoparticles may appear to have a first observable color, and a second different observable color under bright field imaging conditions, as described in www.sigmaaldrich.com / US / en / technical-documents / technical-article / materials-science-and-engineering / biosensors-and-imaging / gold-nanoparticles.

[0047] Polymer / Biomacromolecular Nanoparticles

[0048] In aspects, the nanoparticle is a polymer / biomacromolecule nanoparticle. Polymer / biomacromolecule nanoparticles are made of biocompatible nanomaterials and may be used in combination with other types of materials to improve their biocompatibility or functionality. In aspects, the nanoparticle may be a polymer nanoparticle comprising a polymer selected from poly(D,L-lactic-co-glycolic acid) (PLGA), polystyrene, polyethylene glycol (PEG) covered or PEGylated nanoparticles, polyethylene glycol (PEG), polyethylene glycol)-block-poly(aspartic acid) (PEG-PAA)-coated calcium phosphate, poly-vinyl-chloride (PVC), or combinations thereof. In aspects, the nanoparticle comprises a material that is coated with a polymer selected from poly(D,L-lactic-co-glycolic acid) (PLGA), polystyrene, polyethylene glycol (PEG), poly(ethylene glycol)-block-poly(aspartic acid) (PEG-PAA)-coated calcium phosphate, poly-vinyl-chloride (PVC), or combinations thereof. In aspects, the polymer / biomacromolecule nanoparticle is a lipid. In aspects, the polymer / biomacromolecule nanoparticle is a lipoprotein. In aspects, the polymer / biomacromolecule nanoparticle is a protein condensed nanoparticles made with albumin and oligonucleotides. In aspects, the polymer / biomacromolecule nanoparticle is a nanoparticle containing DNA in addition to inorganic molecules or non-nucleic acid polymers such as polyethylene glycol / DNA nanoparticles, poly(methyl methacrylate) / poly(ethyleneimine)-nanoparticle / pDNA complexes, poly-L-Lysine-DNA complexes, and combinations thereof. In aspects, the polymer / biomacromolecule nanoparticle is a fluorescent polymer nanoparticle. In aspects, the nanoparticle comprises a surface modifier that serves to increase cellular uptake of the nanoparticle. In aspects, the nanoparticle comprises a surface modifier that serves to increase the specificity of the nanoparticle. In aspects, the nanoparticle comprises a surface modifier that serves to increase the efficiency of intracellular targeting or retention of the nanoparticle. In aspects, the nanoparticle comprises a surface modifier that is a peptide. In aspects, the nanoparticle comprises a surface modifier that is a plurality of peptides. In aspects, the nanoparticle comprises a surface modifier that is an antibody. In aspects, the nanoparticle comprises a surface modifier that is a plurality of antibodies. In aspects, the nanoparticle comprises a size-dependent optical property. For example, the size-dependent optical property may be quantum confinement in different semiconductor nanoparticles, an absorbance of surface plasmon resonance in metal (particularly noble metals) nanoparticles, superparamagnetism in magnetic nanoparticles or the like. In some examples, a nanoparticle can comprise a polymeric core material coated or plated with a metallic material (e.g., gold or silver).Color Nanoparticles

[0049] In aspects, the nanoparticle has a size and / or shape that causes or induces the nanoparticle to have a detectable color. As used herein, a nanoparticle “having” a color encompasses a nanoparticle having an “inducible” color. In aspects, the nanoparticle has a shape that causes (induces) the nanoparticle to have a detectable color. For example, the ratio of rod length to diameter on a nanoparticle can cause, induce, or bring about a particular detectable color (e.g., when attached in bulk to a region of a cell). An example of an induced color includes aggregation of the bound nanoparticle which, as a result of the aggregation, results in the ability to observe and detect, and in some instances quantify, the color emitted by the nanoparticle. By modulation of the rod length and / or diameter, a desired color can be obtained. In aspects, the nanoparticle has a size that causes the nanoparticle to have a detectable color. In other aspects, the nanoparticle shape can induce a certain color characteristic in aggregate amounts. That is, a plurality of nanoparticles having a similar shape may cause a certain color in a brightfield imaging environment as a result of how the particles impact light emitted form a light source when aggregated or before the light is conveyed to a camera to capture cell images. Thus, the size and / or shape of the nanoparticle may be preselected to provide a desired color under brightfield conditions for imaging of the cell. The nanoparticles used in the disclosed methods may be selected based on a color; any visible colors may be used with the disclosed methods. In aspects, the nanoparticle is selected based on the color generated by or induced by the nanoparticle when illuminated under brightfield conditions, for example by a light source such as a halogen lamp or light-emitting diode (LED). In aspects, the nanoparticle material is selected based on the color as seen using brightfield imaging, and may include any nanoparticle material as described herein.

[0050] In aspects, the color of the nanoparticle prior to binding to a target antigen is identical to the color as generated by or induced by imaging under brightfield conditions. For example, a nanoparticle may be red and may be visualized as red under brightfield conditions. In aspects, the nanoparticle is orange and may be visualized as orange under brightfield conditions. In aspects, the nanoparticle is yellow and may be visualized as yellow under brightfield conditions. In aspects, the nanoparticle is green and may be visualized as green under brightfield conditions. In aspects, the nanoparticle is blue and may be visualized as blue under brightfield conditions. In aspects, the nanoparticle is purple / violet and may be visualized as purple / violet under brightfield conditions.In aspects, the nanoparticle is grey and may be visualized as grey under brightfield conditions. In aspects, the nanoparticle is brown and may be visualized as brown under brightfield conditions.

[0051] In aspects, the nanoparticle material may be perceived as colorless, but induce a particular color when bonded in aggregate to a cell or cell region (e.g., due to construction, shape, or sizing characteristics of the nanoparticle). In aspects, the nanoparticle material used may be perceived as colorless, but due to the size, a color is detected as a result of the density or aggregation of the nanoparticles on a cell or region of interest of a cell.

[0052] In aspects, a plurality of different nanoparticles, each having a different color (e.g., two or more different colors, or three or more different colors, or four or more different colors, or five or more different colors, or more than six different colors, depending on the number of cell types or cell antigens to be assayed) is used. In aspects, the plurality of different nanoparticles comprise nanoparticles of different materials. In aspects, the plurality of different nanoparticles comprise the same material but each nanoparticle targets a different cell antigen and has a different color to allow correlation of the color to the cell having the target antigen. In aspects, the plurality of different nanoparticles comprise the same material but comprise a different shape (e.g., differences between diameter / width and length dimensions) to induce a different color in a brightfield imaging environment.Methods

[0053] As set forth above, currently available methods for discerning cell types in a sample using imaging may be limited due to the inability to visually distinguish cell types. The disclosed methods allow for the detection and / or characterization of one or more cell types in a fluid sample, for example a biological sample such as blood or serum obtained from an individual for the purpose of diagnosing and / or treating the individual. The disclosed methods provide enhanced detection methods using a nanoparticle conjugated to a binding agent (such as an antibody or antibody fragment) having binding specificity for a target cell antigen. The colors induced by the bound nanoparticles can then be visualized via brightfield methods. The data obtained via the detected nanoparticles can then be used to determine cell type, cell morphology, and / or cell phenotype in a sample based on the detected color indicating specific binding to preselected cell antigens. Such information may then be used for diagnosis, treatment, and / or other monitoring of an individual.

[0054] The disclosed methods may be useful to carrying out a range of different assays for evaluating a fluid sample comprising at least one cell of interest. For example, the methods may be configured to provide “single-plex” methods (in which a single cell type is detected using a single nanoparticle conjugate), multiplex methods (in which multiple nanoparticle conjugates are used to detect multiple cell types), and cell phenotyping methods, in which multiple nanoparticle conjugates are designed for binding to different target cell antigens on the same cell. Each of the methods may be further combined with staining techniques to provide further information about the cells in the sample, as described herein. Non-limiting descriptions of each method is provided below.Single-Plex Methods

[0055] In one aspect, disclosed is a “single-plex” assay in which a single nanoparticle conjugate is used to detect a single type of antigen on a surface of a cell in a fluid sample, whether for determining cell type or disease status. In this aspect, a nanoparticle conjugate having specific binding to a single cell type a detectable color is contacted with a sample suspected of having a cell type of interest. Following an incubation period in which the nanoparticle conjugate binds to cells having the antigen specific to the binding agent of the nanoparticle conjugate, and a subsequent wash step to remove nonspecifically bound nanoparticle conjugate, the nanoparticle conjugate bound to a specific cell marker is detected. Detection of the nanoparticle allows for identification of the specific cell type. For example, in this aspect, a single color nanoparticle comprising a binding agent specific to a unique antigen on a cell surface, is used to detect a single cell type under brightfield conditions. Similarly, the disclosed methods may employ a single-plex assay to determine a disease state, in which a single antigen present on a cell surface is indicative of the presence or absence of a disease. In this aspect, both the presence or absence of binding can be used to obtain information about the cell types in the sample or a disease state of the individual. By way of example, a nanoparticle conjugate comprising an antibody that binds specifically to an antigen unique to the subset of cells to be labeled is contacted with a fluid sample. The nanoparticle conjugate selectively attaches only to cells in the fluid sample having the corresponding target antigen. The nanoparticle conjugate thus labels the specific subset of cells of interest in the fluid sample. Conversely, where no binding is observed, it may be concluded that there are no cell types in the sample having the target antigen.

[0056] In this aspect, the detection of a single color nanoparticle-conjugate via brightfield imaging may be used to provide information about the cell type. For example, the nanoparticleconjugate may comprise a binding agent specific to a target molecule. Exemplary target molecules include CD3, DE4, CD8, CD19, CD14, CD45, CD56, CD80, CD86, CCD40, CD95, and combinations thereof, as described herein.

[0057] FIG. 2 illustrates an embodiment in which a nanoparticle conjugate is used to detect a cell type. Following contact of a sample with the nanoparticle conjugate the fluid sample is subjected to analysis. FIG. 2 depicts an image obtained using the disclosed method. In particular, FIG. 2 shows images of CD34+ cells labeled with nanoparticles having a red color. The red-colored particles are coated with antibodies specific to CD34+ cells. As can be seen in the image in FIG. 2, the color and size of the particles allow the nanoparticle-bound cells to be easily distinguished from non-labeled cells. When the prepared sample fluid is injected into a flow cell, the nanoparticle-labeled cells can be easily distinguished from other cells in the flow path. The method of labeling cells in a fluid sample with nanoparticles prior to flow imaging improves the identification and quantification of particles in the fluid sample, particularly blood samples. As can also be seen in the images, the number of particles attached to each monocyte may vary. Additionally, each image patch can include more than one tagged or untagged cell

[0058] The single-plex method may be used to detect any cell type in a sample for which a target antigen is known. For example, in aspects, the cell type that is detected is a blast cell, wherein the blast cell can be distinguished from the other cells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the blast cell. In aspects, the cell type that is detected is a monocyte, wherein monocyte can be distinguished from the other cells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the monocyte, wherein the nanoparticle conjugate comprises an anti-CD14 antibody. In aspects, the cell type that is detected is a red blood cell, wherein the red blood cell can be distinguished from the other cells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the red blood cell. In aspects, the cell type that is detected is a reticulocyte, wherein the reticulocyte can be distinguished from the other cells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the reticulocyte. In aspects, the cell type that is detected is a nucleated red blood cell, wherein the nucleated red blood cell can be distinguished from the othercells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the nucleated red blood cell. In aspects, the cell type that is detected is an immature red blood cell, wherein the immature red blood cell can be distinguished from the other cells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the immature red blood cell. In aspects, the cell type that is detected is a neutrophil, wherein the neutrophil can be distinguished from the other cells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the neutrophil. In aspects, the cell type that is detected is a lymphocyte, wherein the lymphocyte can be distinguished from the other cells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the lymphocyte. In aspects, the cell type that is detected is a eosinophil, wherein the eosinophil cell can be distinguished from the other cells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the eosinophil. In aspects, the cell type that is detected is a basophil, wherein the basophil can be distinguished from the other cells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the basophil. In aspects, the cell type that is detected is a precursor (immature) white blood cell, wherein the precursor (immature) white blood cell can be distinguished from the other cells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the precursor (immature) white blood cell. In aspects, the cell type that is detected is a lymphoblast, wherein the lymphoblast can be distinguished from the other cells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the lymphoblast. In aspects, the cell type that is detected is a myeloblast, wherein the myeloblast can be distinguished from the other cells in the sample via detection of a color corresponding to a nanoparticle conjugate bound to the myeloblast.

[0059] A multiplex technique, further described herein, refers to a plurality of nanoparticle types used to bond to cells, and comprise a single-cell multiplex and a multiple-cell multiplex. A multiple-cell multiplex is where each of a plurality of nanoparticle types are configured to attach to different cell receptors unique to different cells (e.g., a first nanoparticle type inducing a first color (e.g., red) which binds to specific cell receptors of monocytes, and a second nanoparticle type inducing a second color (e.g., blue) which binds to specific cell receptors of lymphocytes). A single-cell multiplex is wherein each of a plurality of nanoparticle types are configured to attach to different cell receptors of a particular type of cell (e.g., a first nanoparticle type inducing a first color (e.g., red) which binds to a first cell receptor of a monocyte, and a second nanoparticle typeincluding a second color (e.g., blue) which binds to a second cell receptor of a monocyte), so as to induce a plurality of different colors among a particular cell type to aid in analysis / phenotyping. A further example of single cell multiplexing can be used to provide an image comprising a mixture or combination of colors from multiple nanoparticles that have different colors being attached to a single cell, which can then be subjected to spectral analysis to determine the combination of attached nanoparticles.

[0060] In aspects, the multiplex assay creates an image comprising more than one detectable color, in which the multiple colors indicate different markers on a cell surface. Specifically, the more than one color represents more than one different protein that is expressed on the cell surface. The imaging systems detect the total light absorbed by the cell, which is mixture of multiple particles bound to the cell surface. The individual wavelengths can then be extracted and the spectral distribution can be determined, such that the cell surface antigens (e.g., proteins or other target molecule) can be identified. In general, the binding agents conjugated to the nanoparticles comprising antibodies or binding fragments thereof specific for a CD marker. The binding agent(s) are selected on the cell type or disease state to be determined. (See, e.g., Table 1).Multi-Cell Multiplex Assays

[0061] As used herein, the term “multiplex” refers to an assay in which at least two different nanoparticle conjugates employing at least two different nanoparticle are used. In aspects, a plurality of nanoparticle conjugates may be used to detect a plurality of cell types. In aspects, a plurality of nanoparticle conjugates may be used to identify the presence or absence of a disease state. Multiplex methods to detect cells may be “1:1 labeling,” “multi-marker labeling,” and combinations thereof. In 1 : 1 labeling, a single nanoparticle conjugate is used to detect a single cell type. More than one 1:1 labeling event may be used in a single sample for multiplex detection of multiple cell types in the single sample. (For example, the methods may employ more than one nanoparticle type to detect cells in a single sample, e.g., a visualized red nanoparticle indicate a red blood cell and a visualized green nanoparticle indicate a monocyte.) In further aspects, “multimarker” labeling may be used to detect more than one cell type. In this aspect, the multiplex assay may comprise contacting at least two nanoparticle conjugates with a fluid sample, the at least two nanoparticle conjugates having binding agents specific to different antigens on a single cell typeto create a detectable cell signature. For example, referring to Table 1, a combination of three different nanoparticle conjugates, each specific to one of CD3, CD4, or CD8 and comprising three distinguishable nanoparticles can be used to detect a T-lymphoid cell. Where all three nanoparticles are detected on a single cell surface, the cell is identified as a T-lymphoid cell. Both 1 : 1 labeling and multi-marker labeling may be carried out in the same sample. In this aspect, more than one cell type can be detected in a sample by employing more than one nanoparticle conjugate, each comprising a nanoparticle having a different inducible color and a different binding agent specific to a different cell type. In aspects, the nanoparticle conjugate comprises a binding agent specific to one or more than one cell-specific cell surface antigen (e.g., protein or other cell surface target) as described herein, selection of which will be understood by one of ordinary skill in the art. For example, the method may comprise detecting at least two different cell types in a sample using at least two different nanoparticles, the at least two different nanoparticles having different colors when imaged. The at least two different nanoparticles having different colors may comprise different, cell-specific binding agents that may be used to detect and distinguish at least two different cell types in a sample. In aspects, the methods may comprise contacting at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten or more different nanoparticle conjugates with a fluid sample, such as a blood sample, wherein the nanoparticle conjugates comprise different antibodies specific to different antigens representing different cell types. For example, a first nanoparticle may comprise a first antibody specific to a first antigen, the first nanoparticle having an inducible first color that is detectable. A second nanoparticle may comprise a second antibody specific to a second antigen, the second nanoparticle having a second color that is detectable and distinguishable from the other nanoparticles in the sample. A third nanoparticle may comprise a third antibody specific to a third antigen, the third nanoparticle having a third color that is detectable and distinguishable from the other nanoparticles in the sample. A fourth nanoparticle may comprise a fourth antibody specific to a fourth antigen, the fourth nanoparticle having a fourth color that is detectable and distinguishable from the other nanoparticles in the sample. A fifth nanoparticle may comprise a fifth antibody specific to a fifth antigen, the fifth nanoparticle having a fifth color that is detectable and distinguishable from the other nanoparticles in the sample. A sixth nanoparticle may comprise a sixth antibody specific to a sixth antigen, the sixth nanoparticle having a sixth color that is detectable and distinguishable from the other nanoparticles in thesample. A seventh nanoparticle that comprises a seventh antibody specific to a seventh antigen, the seventh nanoparticle having a seventh color that is detectable and distinguishable from the other nanoparticles in the sample. An eighth nanoparticle may comprise a eighth antibody specific to an eighth antigen, the eighth nanoparticle having an eight color that is detectable and distinguishable from the other nanoparticles in the sample. In aspects, the methods may employ more than eight different nanoparticle-antigen-color combinations. In aspects, each of the aforementioned nanoparticle-antigen conjugates may be used in a single assay to determine the various cell types in a sample. The nanoparticle conjugates may be introduced into the sample simultaneously or sequentially.

[0062] Thus, in this aspect, the methods permit the detection of more than one cell type using more than one nanoparticle conjugate in a sample, wherein each nanoparticle conjugate detects a cell type. Non-limiting examples of cell types that can be detected and disease states that can be identified, along with the corresponding cell surface antigens, are shown in Table 1. Further instances of cell types and disease states that can be identified using the disclosed methods will be readily appreciated by one of ordinary skill in the art.

[0063] In addition to the characterizations described above, due to the differences in antigen number on the surface of a cell type, the differential binding of the nanoparticle conjugate to different cell types may further allow for the assessment of the different cell types and subtypes. For example, a cell may have a higher density of a given antigen such that binding of the nanoparticle conjugate to the surface of the cell via that antigen will result in a stronger signal, as compared to a cell type that has a lower density of a given antigen. Cell types may then be distinguished based on the density of the nanoparticle bound to the cell.

[0064] Exemplary cell types that can be detected include one or more of reticulocytes, nucleated red blood cells, immature red blood cells, neutrophils, lymphocytes, eosinophils, basophils, precursor (immature) white blood cells, lymphoblasts, myeloblasts, monocytes, thrombocytes and combinations thereof. Exemplary disease states that can be determined are shown in Table 1.Single-Cell Multiplex

[0065] In aspects, a plurality of nanoparticle conjugates may be used to identify a single cell type or disease state. In one aspect, a multiplex assay for determining a cell type, comprising contacting at least two nanoparticle conjugates with a fluid sample, the at least two nanoparticle conjugates having binding agents specific to different antigens on a single cell type is disclosed. In this aspect, different nanoparticle conjugates may comprise binding agents specific to different cell antigens (e.g., Cluster of Differentiation (CD) glycoproteins) and may be combined in a single assay to determine a cell type based on the binding of the different nanoparticles. The binding of different nanoparticle conjugates to different target antigens on a cell yield images comprising more than one color on a cell, which allows for complex phenotyping based on the displayed color. In one aspect, a method of single cell multiplexing is disclosed in which multiple nanoparticles of different colors are attached to a single cell type, which can create a variation of colors (e.g., brown, off-brown, or shades of a particular color) which can be analyzed via detecting the frequency of the particular colors or shades of colors. In a further aspect, the disclosed methods use different color nanoparticles as described herein, such that the binding of multiple nanoparticle conjugates to a cell forms a distinct color pattern that indicates a cell type or cell phenotype, in contrast to the brown / off-brown / shading resulting from a combination of colors described above. Yet further, the absence of a particular color indicates the absence of a cell antigen, which may further be used to gather information about the cell for phenotyping purposes.

[0066] Referring to FIG. 4, which was described earlier in some detail, nanoparticles will absorb a certain wavelength domain of light resulting in an observable color in abrightfield setting. In some examples, this spectral wavelength analysis can be used to identify the types of nanoparticles bound to a particular cell type - for instance, by identifying multiple peaks in a spectral distribution, or alternatively attuning a resulting observable color of the cell (e.g., brown or off-brown) to the combination of nanoparticle colors that would produce the specific observable color. The spectral wavelength analysis can be used to find the colors of nanoparticle-types bound to the cell, which can be correlated to associated CD markers those particular colored nanoparticles are bound to, which can be used to identify a type of cell being observed. This can further be extended to then identifying particular disease states associated with the type of cell being observed (e.g., if associated with the body’s response to a particular cancer or infection).

[0067] An exemplary multiplex assay may be carried out as follows. A first nanoparticle conjugate having a first color may be used as described above, in which the nanoparticle conjugate has binding specificity to a first cell surface marker on a first cell type. In the same assay, a second nanoparticle conjugate having a second color may be used, wherein the second nanoparticle conjugate has binding specificity to a second cell surface marker on the same (first) cell type. In the same assay, a third nanoparticle conjugate having a third color may be used, wherein the third nanoparticle conjugate has binding specificity to a third cell surface marker on the same (first) cell type. It should be understood that more than three nanoparticle conjugates may be used, for example four, or five, or six, or seven, or eight, nine, ten, or more nanoparticle conjugates, each directed to a different cell antigen. In one aspect, the target antigen is a Cluster of Differentiation (CD) glycoprotein as described herein. Following binding of the more than one nanoparticle conjugate to cells in a sample, the cells are imaged, and the pattern of nanoparticle conjugates bound to a cell type is detecting, and the cell type (or phenotype) is determined based on the color pattern.

[0068] By way of example, a nanoparticle conjugate composition may comprise a red, green and yellow nanoparticle conjugates, each having a different binding agent. Following contact with a fluid sample, one cell type may bind to each of the red nanoparticle, green nanoparticle, and yellow nanoparticle, thus indicating a particular cell type or cell phenotype. Conversely, a different cell may bind to only the red nanoparticle and a green nanoparticle, thus indicating a different cell type or cell phenotype. By assaying for the presence or absence of various colors (indicting binding of various nanoparticles conjugated to different binding agents) a cell type or phenotype can be determined, which may be used for a variety of diagnostic and / or therapeutic purposes.

[0069] In aspects, the multiplex assay may employ a plurality of nanoparticle conjugates having different binding agents to target a specific subset of cells in a fluid sample. For example, a first plurality of nanoparticle conjugates may comprise binding agents that target neutrophils (a first subset of cells), a second plurality of nanoparticle conjugates may comprise binding agents that target basophils (a second subset of cells), a third plurality of nanoparticle conjugates may comprise binding agents that target eosinophils (a third subset of cells), and so on. In aspects, the nanoparticle conjugates may comprise binding agents that target each of a reticulocyte, a nucleated red blood cell, an immature red blood cell, a neutrophil, a lymphocyte, an eosinophil, a basophil,a precursor (immature) white blood cells, a lymphoblast, a myeloblast, a monocyte, a thrombocyte, and combinations thereof. Unidentified cells or cells that do not have known target antigens can be identified by process of elimination. That is, in some aspects, cells with no known target antigens can be identified by tagging all other known cells with nanoparticle conjugates. In some aspects, the particles can tag cells in a fluid sample based on specific characteristics of the cells. For example, the cells may be tagged by size, cytoplasmic characteristics, granularity, nucleation, etc.

[0070] Additional multiplex techniques can combine the single-cell multiplex concept (e.g., to use different nanoparticle induced colors to identify regions of interest of a specific cell to aid in analysis / phenotyping) and the multiple-cell multiplex concept (e.g., to use different nanoparticle induced colors to differentiate between multiple cell types). For instance, a first nanoparticle type and second nanoparticle type induce different colors to differentiate among two different cell types (e.g., induce monocytes to appear red, and lymphocytes to appear blue), while also helping to differentiate a different cell type by inducing different colors within that particular cell type (e.g., identifying blasts by inducing certain regions of the blast to appear as brown and purple). Selection of binding agents to be used with the disclosed methods will be understood by one of ordinary skill in the art. Non-limiting examples of combinations of cell surface antigens that can be used for determining a cell type or disease state are shown in Table 1. For example, referring to Table 1, a combination of three different nanoparticle conjugates, each specific to one of CD3, CD4, or CD8 and comprising three different nanoparticles can be used according to the disclosed methods to determine the presence or absence of a T-lymphoid cell in a sample. Following incubation of the nanoparticle compositions with a sample of interest and removal of nonspecific binding, a cell having all three nanoparticles bound to its surface is identified as a T-lymphoid cell.Further Processing

[0071] Any of the above methods may further comprise contacting the sample with a stain, for example a nuclear stain or other stain that permits visualization of cell morphology. Cell morphology refers to the size, shape, and / or structure of cells, and includes stains for visualization of the cell nucleus. Data obtained by detection of a nanoparticle color may be combined with data obtained from stained cells to further aid in determining cell type and / or phenotype. For example,the specific Cluster of Differentiation (CD) glycoproteins present on the cell (as imaged via the nanoparticle color as described above) can be determined via binding of nanoparticle conjugates as described herein, and such data may be combined with cell morphology information obtained via cell staining to assess one or more of a cell type, cell differentiation stage, cell subtype, and / or cell abnormality. Cell staining may be carried out using methods known in the art. For example, a sample may be contacted with hematoxylin (an acidic dye that targets basic structures) and an eosin counterstain to visualize cytoplasmic contents. In other aspects, the sample may be contacted with a staining reagent selected from one or more of acetocarmine, methylene blue, new methylene blue, crystal violet, eosin, acid fuchsin, safranin, Janus Green B, hematoxylin, Giemsa solution, Wright solution, and Wright-Giemsa solution, Leishman staining solution, Gram staining solution, carbol-fuchsin, Ziehl-Neelsen solution, and combinations thereof. Additional details on staining compounds can be found in US Pat. Nos. 9,279,750 and US Pub. No. US20210108994, the contents of all of which are hereby incorporated by reference in their entirety. In aspects, the sample is contacted first with one or more nanoparticles as described herein, followed by contact with a stain that allows for visualization of cell morphology. In aspects, the sample is contacted first with a stain that allows for visualization of cell morphology followed by one or more nanoparticles as described herein. In aspects, the one or more nanoparticles and stain that allows for visualization of cell morphology are contacted with a sample simultaneously. In aspects, the sample is contacted with a nanoparticle as described herein, followed by stain..Combination Assays

[0072] In aspects, the disclosed methods may comprise a combination of the aforementioned methods. For example, the method may comprise any combination of the following:a single-plex assay, comprising contacting a fluid sample suspecting of containing a cell type of interest with a composition comprising a nanoparticle conjugate specific for a first marker on or in a cell;an assay for detecting a plurality of cell types, comprising contacting a fluid sample suspecting of containing a cell type of interest with a composition comprising at least two nanoparticle conjugate, each comprising a different color, wherein the each of the at leasttwo nanoparticle conjugate are specific for at least two different marker on or in a cell, wherein the at least two nanoparticle conjugate identify different cell types;a multiplex assay, comprising contacting a fluid sample containing a cell type of interest with a composition comprising a plurality of nanoparticle conjugate, the plurality of nanoparticle conjugates being specific for different markers on a single cell type; andan assay for assaying cell morphology, comprising contacting a fluid sample with a stain.Sample Preparation

[0073] Further disclosed are methods of preparing a fluid sample for imaging or flow imaging for carrying out the analytical methods disclosed herein. In aspects, the method for preparing a sample for flow imaging comprises: providing a fluid sample comprising a plurality of cells; mixing the fluid sample with a nanoparticle conjugate or suspension of nanoparticle conjugates to label a cell or subset of cells in the fluid sample; incubating the mixed fluid sample; and processing the mixture in a flow imaging instrument.

[0074] In aspects, the fluid sample to be analyzed may be a blood sample, a urine sample, or any other fluid of interest. Other sample types may include serum, bone marrow, lavage fluid, effusions, exudates, cerebrospinal fluid, pleural fluid, peritoneal fluid, and amniotic fluid. The sample may be prepared by mixing an aliquot of a cell-containing fluid sample, e.g., blood, with a nanoparticle conjugate. In one aspect, a 1-100 (e.g., about 50) microliter aliquot of the cellcontaining fluid is mixed with about 50-300 (e.g., about 200) microliters of the nanoparticle suspension. After mixing the cell-containing fluid sample with the nanoparticle conjugate, the mixture is incubated sufficient to allow binding of the nanoparticle conjugates to the target antigens. During incubation, antibodies on the surface of the particles bind to the epitope of the target antigen of a subset of cells in the fluid. The time and temperature for the incubation will be understood by one of skill in the art. In one aspect, the incubation is carried out prior to contact with a flow imaging instrument. In other aspects, the incubation is carried out inside a flow imaging instrument. In other aspects, the incubation period may be initiated outside of a flow imaging instrument and completed inside a flow imaging instrument. In aspects, a wash step may be carried out to remove nonspecific binding, followed by imaging of the nanoparticle complex bound to thecell for detection of the cell types / cell subtypes of interest. In aspects, the nanoparticle complex bound to the cell for detection of the cell types / cell subtypes of interest without a wash step being carried out prior to the imaging.Binding Agents

[0075] In aspects, the binding agent is an antibody. The antibody may be a monoclonal antibody. In aspects, the antibody is a polyclonal antibody. In aspects, the antibody is a commercial antibody that specifically binds to a target antigen. In yet further aspects, the binding agent is a fragment of an antibody that retains specific binding to target protein / antigen. Thus, as used herein, the term binding agent encompasses an antibody and a binding fragment thereof. The manufacture of antibodies and antibody fragments is understood by one of ordinary skill in the art. Further, commercially available antibodies and antibody fragments directed to an antigen as described herein are available, and may be used with the disclosed methods. The antibody species can be any species, provide the antibody is specific to the antigen, and may be, for example, of mammalian origin, for example a rabbit or mouse antibody.Target Antigens

[0076] Suitable target antigens will be understood by one of ordinary skill in the art. In aspects, the target antigen is an antigen predominantly or exclusively found on a red blood cell. In aspects, the target antigen is an antigen predominantly or exclusively found on a white blood cell. In aspects, the target antigen is an antigen predominantly or exclusively found on a platelet. In aspects, the target antigen is an antigen predominantly or exclusively found on a neutrophil. In aspects, the target antigen is an antigen predominantly or exclusively found on a lymphocyte. In aspects, the target antigen is an antigen predominantly or exclusively found on a monocyte. In aspects, the target antigen is an antigen predominantly or exclusively found on an eosinophil. In aspects, the target antigen is an antigen predominantly or exclusively found on a basophil. In aspects, the target antigen is an antigen predominantly or exclusively found on a reticulocyte. In aspects, the target antigen is an antigen predominantly or exclusively found on a nucleated red blood cell. In aspects, the target antigen is an antigen predominantly or exclusively found on a blast cell.

[0077] Exemplary cell markers include, for example, CD3, found on T cells, CD3 is involved in T cell receptor (TCR) signaling and activation, CD4, primarily expressed on helper T cells, CD4 interacts with major histocompatibility complex class II (MHC-II) molecules on antigen-presenting cells (APCs) to facilitate T cell activation; CD8, expressed on cytotoxic T cells, CD8 binds to MHC-I molecules on target cells and contributes to the killing of infected or abnormal cells; CD19, present on B cells, CD19 plays a role in B cell signaling and activation; CD14, expressed on monocytes, macrophages, and dendritic cells, CD14 acts as a co-receptor for lipopolysaccharide (LPS) and plays a role in innate immune responses; CD45, found on nucleated hematopoietic cells, CD45 is a protein tyrosine phosphatase involved in lymphocyte development and activation; CD56, expressed on natural killer (NK) cells and a subset of T cells, CD56 is involved in NK cell cytotoxicity and immunoregulation; CD80 (B7-1) and CD86 (B7-2), expressed on APCs, CD80 and CD86 are co- stimulatory molecules that interact with CD28 on T cells to provide the "second signal" necessary for T cell activation; CD40, found on APCs, CD40 interacts with CD40 ligand (CD40L) on activated T cells to mediate immune responses, including B cell activation and antibody production; CD95 (Fas), expressed on various immune cells, CD95 is involved in apoptosis (programmed cell death) and immune regulation. In one aspect, the marker is CD34 and is used to assess the hematopoietic stem cell (HSC) numbers in peripheral blood, cord blood, and apheresis specimens used for peripheral blood stem cell transplantation.

[0078] In aspects, the disclosed compositions and methods can be used to identify a cell type in a sample, via binding of a detectable nanoparticle conjugated to a binding agent to a cell. For example, a cell can be identified as one or more of a B-lymphoid cell, a T-lymphoid cell, a myeloid cell, or a plasmacytoid cell. In this aspect, a nanoparticle is conjugated to an antibody specific to a target molecule on the cell. For example, the target molecule can be selected from CD3, CD4, CD8, CDllb, CD14, CD19, CD20, CD33, CD38, CD43, CD117, and CD138. A plurality of antibodies conjugated to antibodies specific to unique cell surface antigens is then incubated with a sample containing cells of interest for a period of time and under conditions sufficient to allow specific binding of the antibodies to the cell surface antigens. Following a wash step, for removal of non-specific antibody binding, the bound antibody-nanoparticle conjugates can be detected. Detection of the color produced by the bound nanoparticle allows for determination of the cell surface antigens. Determining the cell surface antigens allows for determination of the cell type. For example, referring to Table 1, where CD 19 and CD20 cell surface proteins are detected on agiven cell, the cell is identified as a B-lymphoid cell. Alternatively, where CD3, CD4, and CD8 cell surface proteins are detected on a given cell, the cell is identified as a T-lymphoid. Where CD14, CD33, CD43, CDllb, CD117 cell surface proteins are detected on a given cell, the cell is identified as a myeloid cell. Yet further, where CD38, CD138 cell surface proteins are detected on a given cell, the cell is identified as a plasmacytoid cell. One of ordinary skill in the art will be able to readily apply the disclosed methods for determination of a cell type.

[0079] In aspects, the disclosed compositions and methods can be used to identify a disease state in the individual based on the proteins expressed on cells in the individual, via binding of a detectable nanoparticle to a cell. For example, a disease state can be identified based on the presence or absence of certain cell surface proteins expressed on a cell surface. In aspects the disease state is identified by the level of expression of cell surface proteins expressed on a cell surface. In aspects, a statistically significant difference in the level of expression as compared to a control indicates the presence of a disease state. Table 1 provides non-limiting examples of combinations of cell surface proteins that, when detected using the disclosed methods, can be used to identify a disease state in an individual. For example, an individual may be diagnosed as having B-ALL, CLL, or DLBL where CD 19 and CD20 are detected on a cell surface at levels that differ as compared to a control value. One of ordinary skill in the art will be able to readily apply the disclosed methods for determination of a disease state.

[0080] Table 1. Cell Type Identification and Disease State Identification (abnormal cells diagnostic of disease)Disease Cell CD MarkersB-cell Acute Lymphoblastic Leukemia (B-ALL)Chronic Lymphocytic Leukemia (CLL)Diffuse Large B-Cell Lymphoma (DLBCL) B-Lymphoid CD 19, CD20 T-cell Acute Lymphoblastic Leukemia (T-ALL)T-cell lymphomas T-Lymphoid CD3, CD4, CD8 Acute Myeloid Leukemia (AML) / Chronic CD 14, CD33, CD43, Myeloid Leukemia (CML) Myeloid CDllb, CD117Myeloma Plasmacytoid CD38, CD138Conjugation Binding Techniques

[0081] In aspects, the nanoparticles used in the disclosed methods are conjugated to a binding agent, for example an antibody or fragment thereof, to form a “nanoparticle conjugate.” The nanoparticle conjugate may be formed using methods known in the art. Both covalent and non-covalent immobilization modes may be used. Exemplary conjugation methods include, for example, ionic adsorption (non-covalent attachment), covalent binding (including carbodiimide chemistry, maleimide chemistry, and click-chemistry or using adapter molecules such as biotin. Further exemplary methods include directional, covalent conjugation via a linker, such as a dithiol linker between the nanoparticle surface and the non-targeting (Fc) region of the antibody (active conjugation), and passive adsorption of antibodies to the nanoparticle surface (passive conjugation). Commercial covalent conjugation kit such as that provided by Innova Biosciences can further be used. See, for example, Tam JO, et al. A comparison of nanoparticle conjugation strategies in sandwich immunoassays. J Immunoassay Immunochem. 2017;38(4):355-377. doi: 10.1080 / 15321819.2016.1269338.Examples

[0082] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1

[0083] The sample preparation is prepared by mixing whole blood with a suspension of nanoparticles coated with an antibody targeting a specific antigen (nanoparticle conjugates) - in one example, an aliquot of about 200 microliters. A typical incubation time for this mixture canrange from 1 minute to 30 minutes, about 2 to about 5 minutes, or about 2 minutes. In one aspect, the total sample preparation time is 5 minutes or less. The mixture is then processed through a flow imaging instrument, (e.g.. the instrument shown and described in FIG. 1) in which each blood cell is isolated based on a set of physical characteristics. One single patch will contain one cell however in some scenarios (e.g., high cell concentration) a patch may contain more than one cell. In this analysis some nanoparticles may be free floating in mixture or attached to the epitope of the targeted antigen. The identification of nanoparticles attached to cells can be “manually” (by visual inspection) or “automatically” (by image processing) performed. The unique color of nanoparticles (in one example orange or dark orange, though various colors are possible as described in the description herein) makes the detection highly specific since the stained white cells do not contain these colors. The sample preparation may be completely automated.

[0084] Example 2.

[0085] Cell morphology (flow images and / or cell smear images) does not always allow for positive identification of specific cells (like B-cells, T-cells and blasts) which is often required for diagnosis. Positive identification of blood cells can be achieved by using an antibody targeting a specific antigen on the cell membrane and visualizing them in flow imaging as different color cells.Exemplary Combinations

[0086] The following examples relate to various non-exhaustive ways in which the teachings herein may be combined or applied. It should be understood that the following examples are not intended to restrict the coverage of any claims that may be presented at any time in this application or in subsequent filings of this application. No disclaimer is intended. The following examples are being provided for nothing more than merely illustrative purposes. It is contemplated that the various teachings herein may be arranged and applied in numerous other ways. It is also contemplated that some variations may omit certain features referred to in the below examples. Therefore, none of the aspects or features referred to below should be deemed critical unless otherwise explicitly indicated as such at a later date by the inventors or by a successor in interest to the inventors. If any claims are presented in this application or in subsequent filings related tothis application that include additional features beyond those referred to below, those additional features shall not be presumed to have been added for any reason relating to patentability.

[0087] Example 1

[0088] A method for determining a cell type or a disease state based on a cell population in a fluid sample, the method comprising: imaging a subset of labeled cells in the fluid sample, the subset of labeled cells being formed via contacting the fluid sample with a nanoparticle composition comprising nanoparticle conjugated to a binding agent allowing specific binding of the nanoparticle with a cell surface; detecting, in the image, a color of the nanoparticle on the subset of labeled cells; and characterizing the labeled cell based on presence or absence of the color.

[0089] Example 2

[0090] The method of any preceding example, further comprising flowing a sheath fluid along a flowpath of a flowcell; injecting the fluid sample into the flowpath of the sheath fluid within the flowcell; imaging the subset of labeled cell along an imaging path to produce a plurality of images, the imaging comprising detection of a color of the nanoparticle; and analyzing each of the plurality of images to identify labeled cells.

[0091] Example 3

[0092] The method of example 1 or 2, wherein the nanoparticle is detected via brightfield imaging.

[0093] Example 4

[0094] The method of any preceding example, wherein the binding agent is specific to an antigen on the cell surface.

[0095] Example 5

[0096] The method of any preceding example, wherein the nanoparticle is a metal nanoparticle.

[0097] Example 6

[0098] The method of example 5, wherein the metal nanoparticle comprises a metal selected from gold, silver, platinum, palladium, cobalt, iron, copper, tin, tantalum, vanadium, molybdenum, tungsten, osmium, iridium, rhenium, hafnium, thallium, lead, bismuth, gadolinium, dysprosium, holmium, uranium, and combinations thereof.

[0099] Example 7

[0100] The method of example 5 or 6, wherein the nanoparticle is a gold nanoparticle.

[0101] Example 8

[0102] The method of any preceding example wherein the nanoparticle comprises a diameter of from about 0.5 to about 500 nm, or from about 5 to about 400 nm, or from about 10 to about 300 nm, or from about 15 to about 200 nm, or from about 20 to about 100 nm, or from about 30 to about 90 nm, or from about 40 to about 80 nm in diameter.

[0103] Example 9

[0104] The method of example 6, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 350 and 500 nm, and which is detectable as a yellow color.

[0105] Example 10

[0106] The method of example 6, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 500 and 575 nm, and which is detectable as a red color.

[0107] Example 11

[0108] The method of example 6, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 575 and 700 nm, and which is detectable as a blue color.

[0109] Example 12

[0110] The method of any preceding example, wherein the binding agent is specific to a cell surface of a cell type selected from a reticulocyte, a nucleated red blood cell, an immature redblood cell, a neutrophil, a lymphocyte, an eosinophil, a basophil, a precursor (immature) white blood cells, a lymphoblast, a myeloblast, a monocyte, a thrombocyte, and combinations thereof.

[0111] Example 13

[0112] The method of any preceding example, wherein the nanoparticle composition comprises a plurality of nanoparticle conjugates specific to different cell surface antigens and wherein characterizing the subset of labeled cells comprises identifying the cell type based on the presence or absence of a detected color or a unique color signature obtained from the plurality of images.

[0113] Example 14

[0114] The method of any preceding example, wherein the nanoparticle composition comprises a plurality of nanoparticle conjugates specific to different cell surface antigens and wherein characterizing the subset of labeled cells comprises determining a disease status based on the presence or absence of a detected color or a unique color signature obtained from the plurality of images.

[0115] Example 15

[0116] The method of any preceding example, wherein the binding agent is an antibody or binding fragment thereof.

[0117] Example 16

[0118] The method of any preceding example, wherein the binding agent has specific binding to a cluster of differentiation (CD) selected from CD3, DE4, CD8, CD19, CD14, CD45, CD56, CD80, CD86, CCD40, CD95, and combinations thereof.

[0119] Example 17

[0120] The method of any one of examples 2 to 16, wherein the plurality of images comprise at least one color, or at least two colors, or at least three colors.

[0121] Example 18

[0122] A system comprising: one or more processors; and a non-transitory computer readable medium having stored thereon instructions to, when executed using the one or more processors, perform a set of acts comprising: imaging a subset of labeled cells in the fluid sample, the subset of labeled cells being formed via contacting the fluid sample with a nanoparticle composition comprising nanoparticle conjugated to a binding agent allowing specific binding of the nanoparticle with a cell surface; detecting, in the image, a color of the nanoparticle on the subset of labeled cells; and characterizing the labeled cell based on presence or absence of the color.

[0123] Example 19

[0124] The system of example 18, the acts further comprising flowing a sheath fluid along a flowpath of a flowcell; injecting the fluid sample into the flowpath of the sheath fluid within the flowcell; imaging the subset of labeled cell along an imaging path to produce a plurality of images, the imaging comprising detection of a color of the nanoparticle; and analyzing each of the plurality of images to identify labeled cells.

[0125] Example 20

[0126] The system of example 18 or 19, wherein the nanoparticle is detected via brightfield imaging.

[0127] Example 21

[0128] The system of any one of examples 18 through 20, wherein the binding agent is specific to an antigen on the cell surface.

[0129] Example 22

[0130] The system of any one of examples 18 through 21, wherein the nanoparticle is a metal nanoparticle.

[0131] Example 23

[0132] The system of example 22, wherein the metal nanoparticle comprises a metal selected from gold, silver, platinum, palladium, cobalt, iron, copper, tin, tantalum, vanadium, molybdenum,tungsten, osmium, iridium, rhenium, hafnium, thallium, lead, bismuth, gadolinium, dysprosium, holmium, uranium, and combinations thereof.

[0133] Example 24

[0134] The system of example 22 or 23 wherein the nanoparticle is a gold nanoparticle.

[0135] Example 25

[0136] The system of any one of examples 18 through 24, wherein the nanoparticle comprises a diameter of from about 0.5 to about 500 nm, or from about 5 to about 400 nm, or from about 10 to about 300 nm, or from about 15 to about 200 nm, or from about 20 to about 100 nm, or from about 30 to about 90 nm, or from about 40 to about 80 nm in diameter.

[0137] Example 26

[0138] The system of example 23, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 350 and 500 nm, and which is detectable as a yellow color.

[0139] Example 27

[0140] The system of example 23, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 500 and 575 nm, and which is detectable as a red color.

[0141] Example 28

[0142] The system of example 23, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 575 and 700 nm, and which is detectable as a blue color.

[0143] Example 29

[0144] The system of any one of examples 18 through 28, wherein the binding agent is specific to a cell surface of a cell type selected from a reticulocyte, a nucleated red blood cell, an immature red blood cell, a neutrophil, a lymphocyte, an eosinophil, a basophil, a precursor (immature) white blood cells, a lymphoblast, a myeloblast, a monocyte, a thrombocyte, and combinations thereof.

[0145] Example 30

[0146] The system of any one of examples 18 through 29, wherein the nanoparticle composition comprises a plurality of nanoparticle conjugates specific to different cell surface antigens and wherein characterizing the subset of labeled cells comprises identifying the cell type based on the presence or absence of a detected color or a unique color signature obtained from the plurality of images.

[0147] Example 31

[0148] The system of any one of examples 18 through 30, wherein the nanoparticle composition comprises a plurality of nanoparticle conjugates specific to different cell surface antigens and wherein characterizing the subset of labeled cells comprises determining a disease status based on the presence or absence of a detected color or a unique color signature obtained from the plurality of images.

[0149] Example 32

[0150] The system of any one of examples 18 through 31, wherein the binding agent is an antibody or binding fragment thereof.

[0151] Example 33

[0152] The system of any one of examples 18 through 32, wherein the binding agent has specific binding to a cluster of differentiation (CD) selected from CD3, DE4, CD8, CD19, CD14, CD45, CD56, CD80, CD86, CCD40, CD95, and combinations thereof.

[0153] Example 34

[0154] The system of any one of examples 18 through 33, wherein the plurality of images comprise at least one color, or at least two colors, or at least three colors.

[0155] Example 35

[0156] A method comprising performing the set of acts the instructions stored on the non-transitory computer readable medium of the system of any of examples 20-36 are to perform when executed.

[0157] Example 36

[0158] An analyzer comprising a flowcell, a camera, one or more processors, and a non-transitory computer readable medium having instructions to perform the set of acts the instructions stored on the non-transitory computer readable medium of the system of any of examples 20-34 are to perform when executed.

[0159] Example 37

[0160] The system of example 36, the acts further comprising flowing a sheath fluid along a flowpath of a flowcell; injecting the fluid sample into the flowpath of the sheath fluid within the flowcell; imaging the subset of labeled cell along an imaging path to produce a plurality of images, the imaging comprising detection of a color of the nanoparticle; and analyzing each of the plurality of images to identify labeled cells.

[0161] Example 38

[0162] The system of example 36 or 37, wherein the nanoparticle is detected via brightfield imaging.

[0163] Example 39

[0164] The system of any one of examples 36 through 38, wherein the binding agent is specific to an antigen on the cell surface.

[0165] Example 40

[0166] The system of any one of examples 36 through 39, wherein the nanoparticle is a metal nanoparticle.

[0167] Example 41

[0168] The system of example 40, wherein the metal nanoparticle comprises a metal selected from gold, silver, platinum, palladium, cobalt, iron, copper, tin, tantalum, vanadium, molybdenum, tungsten, osmium, iridium, rhenium, hafnium, thallium, lead, bismuth, gadolinium, dysprosium, holmium, uranium, and combinations thereof.

[0169] Example 42

[0170] The system of any one of examples 36 through 41 wherein the nanoparticle is a gold nanoparticle.

[0171] Example 43

[0172] The system of any one of examples 36 through 42, wherein the nanoparticle comprises a diameter of from about 0.5 to about 500 nm, or from about 5 to about 400 nm, or from about 10 to about 300 nm, or from about 15 to about 200 nm, or from about 20 to about 100 nm, or from about 30 to about 90 nm, or from about 40 to about 80 nm in diameter.

[0173] Example 44

[0174] The system of example 41, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 350 and 500 nm, and which is detectable as a yellow color.

[0175] Example 45

[0176] The system of example 41, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 500 and 575 nm, and which is detectable as a red color.

[0177] Example 46

[0178] The system of example 41, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 575 and 700 nm, and which is detectable as a blue color.

[0179] Example 47

[0180] The system of any one of examples 36 through 46, wherein the binding agent is specific to a cell surface of a cell type selected from a reticulocyte, a nucleated red blood cell, an immature red blood cell, a neutrophil, a lymphocyte, an eosinophil, a basophil, a precursor (immature) white blood cells, a lymphoblast, a myeloblast, a monocyte, a thrombocyte, and combinations thereof.

[0181] Example 48

[0182] The system of any one of examples 36 through 47, wherein the nanoparticle composition comprises a plurality of nanoparticle conjugates specific to different cell surface antigens and wherein characterizing the subset of labeled cells comprises identifying the cell type based on the presence or absence of a detected color or a unique color signature obtained from the plurality of images.

[0183] Example 49

[0184] The system of any one of examples 36 through 48, wherein the nanoparticle composition comprises a plurality of nanoparticle conjugates specific to different cell surface antigens and wherein characterizing the subset of labeled cells comprises determining a disease status based on the presence or absence of a detected color or a unique color signature obtained from the plurality of images.

[0185] Example 50

[0186] The system of any one of examples 36 through 49, wherein the binding agent is an antibody or binding fragment thereof.

[0187] Example 51

[0188] The system of any one of examples 18 through 50, wherein the binding agent has specific binding to a cluster of differentiation (CD) selected from CD3, DE4, CD8, CD19, CD14, CD45, CD56, CD80, CD86, CCD40, CD95, and combinations thereof.

[0189] Example 52

[0190] The system of any one of examples 36 through 51, wherein the plurality of images comprise at least one color, or at least two colors, or at least three colors.Conclusion

[0191] All percentages and ratios are calculated by weight unless otherwise indicated.

[0192] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0193] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0194] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”

[0195] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUBMED, PUBCHEM, NCBI, UNTPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0196] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

CLAIMSWhat is claimed is:

1. A method for determining a cell type or a disease state based on a cell population in a fluid sample, the method comprising:imaging a subset of labeled cells in the fluid sample, the subset of labeled cells being formed via contacting the fluid sample with a nanoparticle composition comprising nanoparticle conjugated to a binding agent allowing specific binding of the nanoparticle with a cell surface;detecting, in the image, a color of the nanoparticle on the subset of labeled cells; andcharacterizing the labeled cell based on presence or absence of the color.

2. The method of any preceding claim, further comprisingflowing a sheath fluid along a flowpath of a flowcell;injecting the fluid sample into the flowpath of the sheath fluid within the flowcell;imaging the subset of labeled cell along an imaging path to produce a plurality of images, the imaging comprising detection of a color of the nanoparticle; andanalyzing each of the plurality of images to identify labeled cells.

3. The method of claim 1 or 2, wherein the nanoparticle is detected via brightfield imaging.

4. The method of any preceding claim, wherein the binding agent is specific to an antigen on the cell surface.

5. The method of any preceding claim, wherein the nanoparticle is a metal nanoparticle.

6. The method of claim 5, wherein the metal nanoparticle comprises a metal selected from gold, silver, platinum, palladium, cobalt, iron, copper, tin, tantalum, vanadium, molybdenum,tungsten, osmium, iridium, rhenium, hafnium, thallium, lead, bismuth, gadolinium, dysprosium, holmium, uranium, and combinations thereof.

7. The method of claim 5 or 6, wherein the nanoparticle is a gold nanoparticle.

8. The method of any preceding claim wherein the nanoparticle comprises a diameter of from about 0.5 to about 500 nm, or from about 5 to about 400 nm, or from about 10 to about 300 nm, or from about 15 to about 200 nm, or from about 20 to about 100 nm, or from about 30 to about 90 nm, or from about 40 to about 80 nm in diameter.

9. The method of claim 8, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 350 and 500 nm, and which is detectable as a yellow color.

10. The method of claim 6, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 500 and 575 nm, and which is detectable as a red color.

11. The method of claim 6, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 575 and 700 nm, and which is detectable as a blue color.

12. The method of any preceding claim, wherein the binding agent is specific to a cell surface of a cell type selected from a reticulocyte, a nucleated red blood cell, an immature red blood cell, a neutrophil, a lymphocyte, an eosinophil, a basophil, a precursor (immature) white blood cells, a lymphoblast, a myeloblast, a monocyte, a thrombocyte, and combinations thereof.

13. The method of any preceding claim, wherein the nanoparticle composition comprises a plurality of nanoparticle conjugates specific to different cell surface antigens and wherein characterizing the subset of labeled cells comprises identifying the cell type based on the presence or absence of a detected color or a unique color signature obtained from the plurality of images.

14. The method of any preceding claim, wherein the nanoparticle composition comprises a plurality of nanoparticle conjugates specific to different cell surface antigens and wherein characterizing the subset of labeled cells comprises determining a disease status based on the presence or absence of a detected color or a unique color signature obtained from the plurality of images.

15. The method of any preceding claim, wherein the binding agent is an antibody or binding fragment thereof.

16. The method of any preceding claim, wherein the binding agent has specific binding to a cluster of differentiation (CD) selected from CD3, DE4, CD8, CD19, CD14, CD45, CD56, CD80, CD86, CCD40, CD95, and combinations thereof.

17. The method of any one of claims 2 to 16, wherein the plurality of images comprise at least one color, or at least two colors, or at least three colors.

18. A system comprising:one or more processors; anda non-transitory computer readable medium having stored thereon instructions to, when executed using the one or more processors, perform a set of acts comprising:imaging a subset of labeled cells in a fluid sample, the subset of labeled cells being formed via contacting the fluid sample with a nanoparticle composition comprising nanoparticle conjugated to a binding agent allowing specific binding of the nanoparticle with a cell surface;detecting, in the image, a color of the nanoparticle on the subset of labeled cells; andcharacterizing the labeled cell based on presence or absence of the color.

19. The system of claim 18, the acts further comprisingflowing a sheath fluid along a flowpath of a flowcell;injecting the fluid sample into the flowpath of the sheath fluid within the flowcell;imaging the subset of labeled cell along an imaging path to produce a plurality of images, the imaging comprising detection of a color of the nanoparticle; andanalyzing each of the plurality of images to identify labeled cells.

20. The system of claim 18 or 19, wherein the nanoparticle is detected via brightfield imaging.

21. The system of any one of claims 18 through 20, wherein the binding agent is specific to an antigen on the cell surface.

22. The system of any one of claims 18 through 21, wherein the nanoparticle is a metal nanoparticle.

23. The system of claim 22, wherein the metal nanoparticle comprises a metal selected from gold, silver, platinum, palladium, cobalt, iron, copper, tin, tantalum, vanadium, molybdenum, tungsten, osmium, iridium, rhenium, hafnium, thallium, lead, bismuth, gadolinium, dysprosium, holmium, uranium, and combinations thereof.

24. The system of claim 22 or 23 wherein the nanoparticle is a gold nanoparticle.

25. The system of any one of claims 18 through 24, wherein the nanoparticle comprises a diameter of from about 0.5 to about 500 nm, or from about 5 to about 400 nm, or from about 10 to about 300 nm, or from about 15 to about 200 nm, or from about 20 to about 100 nm, or from about 30 to about 90 nm, or from about 40 to about 80 nm in diameter.

26. The system of claim 23, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 350 and 500 nm, and which is detectable as a yellow color.

27. The system of claim 23, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 500 and 575 nm, and which is detectable as a red color.

28. The system of claim 23, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 575 and 700 nm, and which is detectable as a blue color.

29. The system of any one of claims 18 through 28, wherein the binding agent is specific to a cell surface of a cell type selected from a reticulocyte, a nucleated red blood cell, an immature red blood cell, a neutrophil, a lymphocyte, an eosinophil, a basophil, a precursor (immature) white blood cells, a lymphoblast, a myeloblast, a monocyte, a thrombocyte, and combinations thereof.

30. The system of any one of claims 18 through 29, wherein the nanoparticle composition comprises a plurality of nanoparticle conjugates specific to different cell surface antigens and wherein characterizing the subset of labeled cells comprises identifying a cell type based on thepresence or absence of a detected color or a unique color signature obtained from the plurality of images.

31. The system of any one of claims 18 through 30, wherein the nanoparticle composition comprises a plurality of nanoparticle conjugates specific to different cell surface antigens and wherein characterizing the subset of labeled cells comprises determining a disease status based on the presence or absence of a detected color or a unique color signature obtained from the plurality of images.

32. The system of any one of claims 18 through 31, wherein the binding agent is an antibody or binding fragment thereof.

33. The system of any one of claims 18 through 32, wherein the binding agent has specific binding to a cluster of differentiation (CD) selected from CD3, DE4, CD8, CD19, CD14, CD45, CD56, CD80, CD86, CCD40, CD95, and combinations thereof.

34. The system of any one of claims 18 through 33, wherein the plurality of images comprise at least one color, or at least two colors, or at least three colors.

35. A method comprising performing the set of acts the instructions stored on the non-transitory computer readable medium of the system of any of claims 20-34 are to perform when executed.

36. An analyzer comprising a flowcell, a camera, one or more processors, and a non-transitory computer readable medium having instructions to perform the set of acts the instructions stored on the non-transitory computer readable medium of the system of any of claims 20-34 are to perform when executed.

37. The system of claim 36, the acts further comprisingflowing a sheath fluid along a flowpath of a flowcell;injecting the fluid sample into the flowpath of the sheath fluid within the flowcell;imaging the subset of labeled cell along an imaging path to produce a plurality of images, the imaging comprising detection of a color of the nanoparticle; andanalyzing each of the plurality of images to identify labeled cells.

38. The system of claim 36 or 37, wherein the nanoparticle is detected via brightfield imaging.

39. The system of any one of claims 36 through 38, wherein the binding agent is specific to the cell surface.

40. The system of any one of claims 36 through 39, wherein the nanoparticle is a metal nanoparticle.

41. The system of claim 40, wherein the metal nanoparticle comprises a metal selected from gold, silver, platinum, palladium, cobalt, iron, copper, tin, tantalum, vanadium, molybdenum, tungsten, osmium, iridium, rhenium, hafnium, thallium, lead, bismuth, gadolinium, dysprosium, holmium, uranium, and combinations thereof.

42. The system of any one of claims 36 through 41 wherein the nanoparticle is a gold nanoparticle.

43. The system of any one of claims 36 through 42, wherein the nanoparticle comprises a diameter of from about 0.5 to about 500 nm, or from about 5 to about 400 nm, or from about 10 to about 300 nm, or from about 15 to about 200 nm, or from about 20 to about 100 nm, or from about 30 to about 90 nm, or from about 40 to about 80 nm in diameter.

44. The system of claim 41, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 350 and 500 nm, and which is detectable as a yellow color.

45. The system of claim 41, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 500 and 575 nm, and which is detectable as a red color.

46. The system of claim 41, wherein the nanoparticle is a gold nanoparticle that emits a wavelength between 575 and 700 nm, and which is detectable as a blue color.

47. The system of any one of claims 36 through 46, wherein the binding agent is specific to a cell surface of a cell type selected from a reticulocyte, a nucleated red blood cell, an immature red blood cell, a neutrophil, a lymphocyte, an eosinophil, a basophil, a precursor (immature) white blood cells, a lymphoblast, a myeloblast, a monocyte, a thrombocyte, and combinations thereof.

48. The system of any one of claims 36 through 47, wherein the nanoparticle composition comprises a plurality of nanoparticle conjugates specific to different cell surface antigens and wherein characterizing the subset of labeled cells comprises identifying a cell type based on the presence or absence of a detected color or a unique color signature obtained from the plurality of images.

49. The system of any one of claims 36 through 48, wherein the nanoparticle composition comprises a plurality of nanoparticle conjugates specific to different cell surface antigens and wherein characterizing the subset of labeled cells comprises determining a disease status based on the presence or absence of a detected color or a unique color signature obtained from the plurality of images.

50. The system of any one of claims 36 through 49, wherein the binding agent is an antibody or binding fragment thereof.

51. The system of any one of claims 18 through 50, wherein the binding agent has specific binding to a cluster of differentiation (CD) selected from CD3, DE4, CD8, CD19, CD14, CD45, CD56, CD80, CD86, CCD40, CD95, and combinations thereof.

52. The system of any one of claims 36 through 51, wherein the plurality of images comprise at least one color, or at least two colors, or at least three colors.

Images