Engineered particles as erythrocyte mimetics for hematology and compositions containing engineered particles

Abstract

Provided herein are red blood cell control compositions comprising one or more populations of cleavable hydrogel particles, the cleavable hydrogel particles having an impedance substantially similar to that of human red blood cells having an average diameter; and a population of hemoglobin molecules or a population of dye molecules having substantially similar absorbance to hemoglobin; wherein (i) and / or (ii) are present in an amount corresponding to a normal blood sample or disease state, or (i) and (ii) are present in a ratio corresponding to a normal blood sample or disease state.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 338,719, filed on May 5, 2022, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] The present disclosure generally relates to red blood cell control compositions and their use for evaluating red blood cell samples. The present disclosure also relates to synthetic whole blood sample controls comprising both a red blood cell control composition and a white blood cell control composition. Background Art

[0003] The changes in the morphology and physiological characteristics of red blood cells in the patient's blood provide valuable information about the pathological conditions of many specific types of red blood cell disorders or anemias. When diagnosing such disorders, the mean cell hemoglobin concentration (MCHC) and mean cell volume (MCV) can be measured to provide valuable understanding of the patient's condition. This information can be combined with the microscope evaluation of the distribution of the size, shape and color of the red blood cells in the stained blood smears performed by trained hematologists and other biochemical tests. The refractive index changes of individual red blood cells are highly correlated with their hemoglobin concentration, and this information can be combined with size measurements to provide diagnostic value. For example, in microcytic anemia, the size of the red blood cells and therefore also the MCV significantly reduce, but the optical density (related to the refractive index) and MCHC increase. In megaloblastic anemia, both size (macroerythrocytes) and MCHC increase.

[0004] Changes in the characteristics or number of reticulocytes in a patient's blood can indicate that the patient's bone marrow is producing red blood cells and can help diagnose a variety of conditions, such as anemia or bone marrow failure. Detecting changes in the characteristics or number of platelets or thrombocytes that assist in the clotting system can help diagnose thrombocythaemia, reactive thrombocythaemia, thrombocytopenia, and platelet dysfunction.

[0005] Given the above, the ability to identify changes in the morphology and physiologic characteristics of red blood cells, reticulocytes, platelets, and other blood components is critical to patient evaluation and successful outcomes.

[0006] Flow cytometry is a technique that allows rapid separation, counting and characterization of individual cells (such as red blood cells), and is commonly used in clinical and laboratory settings for a variety of applications. Flow cytometry based on optics relies on directing a beam of light onto a fluid stream that is focused hydrodynamically. Multiple detectors are then aimed at the point where the stream passes through the beam: one detector is aligned with the beam (forward scatter or FSC) and several detectors are perpendicular to the beam (side scatter or SSC). FSC is related to cell volume, while SSC depends on the internal complexity of the particles (e.g., the shape of the nucleus, the amount and type of cytoplasmic particles, or membrane roughness). Due to these correlations, different specific cell types exhibit different FSC and SSC, allowing cell types to be distinguished.

[0007] However, optical-based flow cytometry is an expensive technology that requires the purchase of expensive reagents. Therefore, simpler methods (such as impedance-based methods) are more commonly used as the main measurement basis in clinical environments. The method based on resistance is derived from the Coulter principle. In such a method based on resistance, whole blood can pass through a hole as narrow as a single cell can pass between two electrodes at a time. The impedance between the electrodes changes as the cell passes therethrough, and is proportional to the cell volume, thereby allowing cell counting and evaluation (e.g., volume measurement). Although it is not possible to determine the type of granular leukocytes of similar size, the impedance-based method can return a complete blood cell count, can determine the three classifications of leukocytes, and can distinguish between, for example, red blood cells, leukocytes, and platelets. Due to its robustness and applicability to common clinical problems, the method based on resistance is widely implemented in nursing, and is used as the technical basis of almost every hematology analyzer on the market (e.g., DxH900, DxH690 analyzer (Beckman Coulter, Inc.), XN-1000, XN-L analyzer (Sysmex, Inc.)).

[0008] However, the ability to identify and measure specific cell types (such as red blood cells, reticulocytes and platelets) depends on the appropriate calibration of the measuring instrument. In the case of, for example, a hematology analyzer, calibration depends on the use of purified cells of the target cell type. Obtaining these purified cells may require expensive, laborious procedures that are prone to batch-to-batch variation. These purified cells can also be amplified with cells obtained from endangered species such as alligators and sharks. Therefore, there is a need in the art for synthetic compositions with adjustable optical and electrical properties that can simulate red blood cells, reticulocytes and platelets in hematology analyzers. Summary of the invention

[0009] In embodiments, the present disclosure relates to a red blood cell control composition, which comprises a population of engineered particles (e.g., hydrogels) and a population of hemoglobin or hemoglobin-like molecules. In embodiments, the hemoglobin-like molecules include, for example, heme, dyes (e.g., having an absorbance substantially similar to the absorbance of hemoglobin), or oxygen-carrying molecules or oxygen-transporting molecules. In specific embodiments, the hemoglobin-like molecules are dyes. Such hemoglobin-like molecules can produce a spectral response substantially similar to the spectral response of hemoglobin. In embodiments, each engineered particle (e.g., hydrogel) comprises, for example, at least one hemoglobin or hemoglobin-like molecule encapsulated in the hydrogel or conjugated to the hydrogel. In embodiments, the population of hemoglobin or hemoglobin-like molecules is independent of (not conjugated to or not encapsulated in) the engineered particles (hydrogels). In embodiments, the engineered particles (e.g., hydrogels) may have at least one representative feature substantially similar to the corresponding features of red blood cells. In embodiments, the at least one representative feature is one or more of optical features and morphological features. In embodiments, the morphological feature is one or more of diameter and volume. In embodiments, the one or more hemoglobin or hemoglobin-like molecules are encapsulated in the engineered particles, or the one or more hemoglobin or hemoglobin-like molecules are bound to the surface of the engineered particles (e.g., hydrogel). In embodiments, the engineered particles (e.g., hydrogel) are degradable. In embodiments, the engineered particles (e.g., hydrogel) can be lysed by hematological lysis buffer. In embodiments, at least one representative feature is determined by pore-based technology, image-based technology, and / or waveform-based technology. In embodiments, the average diameter of each engineered particle is between about 1 μm and about 20 μm. In embodiments, the colony of the engineered particles further comprises a plurality of engineered particle subgroups, each of which has at least one representative feature, and the at least one representative feature includes one or more of optical features and morphological features, wherein the morphological features include at least one of diameter and volume. In embodiments, the hemoglobin molecule is a human, mouse, cattle, sheep, bird, dog, cat, pig or plant hemoglobin molecule.

[0010] In an embodiment, the present disclosure provides a red blood cell control composition comprising: (i) a population of one or more cleavable hydrogel particles (e.g., lysable in the presence of a lysis buffer) having an impedance substantially similar to the impedance of a human red blood cell having an average diameter; and (ii) a population of hemoglobin molecules or a population of dye molecules having an absorbance substantially similar to that of hemoglobin; wherein (i) and / or (ii) are present in an amount corresponding to a normal blood sample or a disease state, or (i) and (ii) are present in a ratio corresponding to a normal blood sample or a disease state.

[0011] In embodiments, the colony of the hemoglobin molecules or the colony of the dye molecules are encapsulated in the one or more cleavable hydrogel colonies. In embodiments, at least one hemoglobin molecule or dye molecule is encapsulated in each cleavable hydrogel. In embodiments, the colony of the hemoglobin molecules or the colony of the dye molecules are attached to the one or more cleavable hydrogel colonies. In embodiments, at least one hemoglobin molecule or dye molecule is attached to each cleavable hydrogel. In embodiments, at least one hemoglobin molecule or dye molecule is covalently attached to each cleavable hydrogel. In embodiments, at least one hemoglobin molecule or dye molecule is non-covalently attached to each cleavable hydrogel (e.g., by biotin / streptavidin interaction). In embodiments, at least one hemoglobin molecule or dye molecule is not encapsulated in each cleavable hydrogel or is not attached to each cleavable hydrogel. In embodiments, the composition is a solution, and the colony of the hemoglobin molecules or the colony of the dye molecules is suspended in the solution, and the one or more cleavable hydrogel particle colonies are suspended in the solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A Optical properties of the disclosed engineered particles (A) compared to polystyrene beads (B) are shown. Figure 1B Demonstrated Coulter's Principle.

[0013] Figure 2 A process for producing the engineered particles of the present disclosure is depicted.

[0014] Figure 3A-3C Bright field and fluorescent images of the engineered particles of the present disclosure are provided.

[0015] Figure 4 One embodiment of engineering parameter adjustments to match and / or mimic desired cell population metrics is shown.

[0016] Figure 5 and Figure 6is a diagram showing an embodiment of how forward scatter, side scatter, and surface properties of engineered particles can be tuned.

[0017] Figure 7A-7D Including a graphical representation of red blood cells (RBC) as characterized by the Coulter principle. Actual RBC is shown on the left (A), wherein the peak count number is shown at a volume (fL) corresponding to about 100 μm. (B), (C) and (D) show engineered particles with controlled Coulter volume as measured by the Coulter principle. (B) shows an engineered particle (or RBC mimic) with a Coulter volume corresponding to a diameter between 3 μm and 8 μm. (C) shows an engineered particle with a Coulter volume corresponding to a diameter of about 12 μm but with a low polymer weight percentage. On the contrary, (D) shows an engineered particle with a Coulter volume corresponding to a diameter of about 12 μm but with a high polymer weight percentage. In a variation, for each of (C) and (D), the Coulter volume can be considered as a sense volume, because the actual diameter of each of these engineered particles can be different according to the porosity of the engineered particles, and the porosity is indicated by the weight percentage of the polymer.

[0018] Figure 8A-8B Embodiments of red blood cell control compositions that mimic certain diseases are shown. Specifically, 5 μm hydrogel particles are microcytosis mimics; 10 μm hydrogel particles are macrocytosis mimics; and 8.5 μm hydrogel particles with normal RBC MCV but high RDW are anisocytosis mimics.

[0019] Fig.9A Shown is the generation of different concentrations of human HGB mimetic using the red dye Allura Red. Fig. 9B The use of bovine HGB to produce different concentrations of human HGB mimics is shown. The box shows the normal level of human HGB. Higher concentrations of red dye or bovine HGB can be used as a hyperhemoglobin mimic; lower concentrations of red dye or bovine HGB can be used as a hypohemoglobin or anemia mimic.

[0020] Fig.10 It was shown that the lysis buffer of the red blood cell control composition did not affect the whole blood measurement of the hematology analyzer. DETAILED DESCRIPTION

[0021] A red blood cell control composition is described herein. The red blood cell control composition is engineered to provide absorbance and impedance characteristics substantially similar to those of a human whole blood sample when measured using a hematology analyzer. To achieve this, the composition comprises (i) one or more cleavable hydrogel particle populations having an impedance substantially similar to the impedance of a human red blood cell having an average diameter; and (ii) a population of hemoglobin molecules or a population of dye molecules having an absorbance substantially similar to that of hemoglobin. The one or more cleavable hydrogel particle populations may be interchangeably referred to herein as red blood cell mimics or RBC mimics, the hydrogel particles having an impedance substantially similar to the impedance of a human red blood cell having an average diameter.

[0022] Advantageously, the red blood cell control composition can be processed by a hematology analyzer in the same manner as a human whole blood sample without interfering with the hematology analyzer, so the red blood cell control composition can be used as a control for a hematology analyzer. For example, in an embodiment, the size (e.g., based on forward scattering), complexity (e.g., based on side scattering) and quantity (e.g., based on impedance) of synthetic red blood cells can be measured. In an embodiment, the red blood cell control composition can be combined with synthetic white blood cells (e.g., as described in US10,753,846, which is incorporated by reference in its entirety) to form a synthetic whole blood sample. The synthetic whole blood sample can then be introduced into a hematology analyzer, and the size, complexity and quantity of synthetic red blood cells and white blood cells can be measured. In an embodiment, the synthetic red blood cells of a synthetic whole blood sample can be cracked so that the size, complexity and quantity of synthetic white blood cells can be measured. In an embodiment, absorbance can be measured with the amount of quantitative hemoglobin or dye.

[0023] The red blood cell control composition can be adjusted to match the impedance and absorption of the target red blood cell composition. For example, in embodiments, the red blood cell control composition can be adjusted to be substantially similar to a blood sample from a normal (healthy) subject, a blood sample from an anemic subject, a blood sample from a subject suffering from anisocytosis or any other disease characterized by abnormal hemoglobin levels or red blood cell characteristics (e.g., size, complexity, and / or number). In embodiments, the subject is a human subject. definition

[0024] Unless otherwise defined herein, the technical and scientific terms used in this specification have the meanings commonly understood by those of ordinary skill in the art. For the purpose of interpreting this specification, the following description of the terms will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa, unless the content clearly dictates otherwise. In the event of any conflict between any description of the terms set forth and any document incorporated herein by reference, the description of the terms set forth below shall prevail.

[0025] The indefinite articles “a” and “an” and the definite article “the” are intended to include both the singular and the plural, unless the context in which they are used clearly indicates otherwise.

[0026] “At least one” and “one or more” are used interchangeably to mean that an item may contain one or more than one of the listed elements.

[0027] Unless otherwise indicated, all numbers expressing quantities of ingredients, ratios and numerical properties, reaction conditions and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about".

[0028] As used herein, the term "about" with respect to a number or numerical range should be understood to mean the stated number and the number + / - 10% thereof, or, for values ​​listed in a range, 10% below the listed lower limit and 10% above the listed upper limit.

[0029] As used herein, the terms "or" and "and / or" include any and all combinations of one or more of the associated listed items.

[0030] As used herein, the terms "including," "includes," "included" and other forms are not limiting.

[0031] As used herein, the term "comprising" and its grammatical equivalents specify the presence of stated features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0032] As used herein, the term "normal" describes a subject, or a biological sample from such a subject, that is healthy and not suffering from a disease or disorder. Cell Counter

[0033] Regarding optical-based cell counters, e.g. Figure 1AAs shown, side scatter provides a general measure of cell complexity, while forward scatter provides a measure of granularity. The two most important passive optical measurements used in optical-based flow cytometers are FSC (forward scatter) and SSC (side scatter), which measure the size and complexity of the target, respectively. Currently, due to these limitations of polystyrene, users must rely on purified cell lines to calibrate fluorescence intensity, inter-laser delay, sorting delay, size, and cell complexity for experiments. This is a long and labor-intensive process that significantly increases the cost of optical-based flow cytometry validation and research processes. More importantly, these calibration cell lines introduce biological variations that lead to differences in data interpretation.

[0034] Resistor-based cell counters based on the Coulter principle (e.g. Figure 1B ) utilizes two chambers separated by a microchannel, each of which contains an electrolyte solution. When particles or cells containing a fluid are drawn through the microchannel between the two separated chambers, each particle causes a brief change in the resistance of the liquid. More information on impedance-based cell counters can be found at www.beckman.com / resources / technologies / coulter-principle / coulter-principle-short-c ourse-chapter-1 , which is incorporated by reference in its entirety for all purposes.

[0035] In other words, the particles pulled through the microchannel while generating an electric current produce an impedance change proportional to the volume of the particles passing through the microchannel. This impedance pulse originates from the displacement of the electrolyte caused by the particles. Cells, as particles with poor conductivity, change the effective cross-section of the conductive microchannel. If the conductivity of these particles is lower than the surrounding liquid medium, the resistance through the channel increases, resulting in a temporary decrease in the current through the microchannel. By monitoring such pulses in the current, the number of particles for a given volume of fluid can be counted. In addition, the magnitude of the current change is related to the size of the particles, making it possible to measure the particle size distribution, which can be associated with the mobility, surface charge and concentration of the particles.

[0036] In any flow cytometry method, calibration is critical for accurate performance. The disclosed engineered particles exhibit tuned properties and are suitable for use as calibration reagents for a range of mammalian or bacterial cell types, including erythrocytes.

[0037] In an embodiment, a composition comprising a plurality of engineered particles is provided, wherein each of the plurality of engineered particles has one or more properties substantially similar to one or more properties of a target cell. In an embodiment, each of the engineered particles of the plurality of engineered particles is synthesized by polymerizing one or more monomers (i.e., to form a homopolymer or copolymer). As discussed further below, the use of bifunctional monomers allows for further derivatization of the hydrogel, for example, with a fluorescent dye, an encapsulated protein, a cell surface marker or an epitope binding fragment thereof, or a combination thereof. Examples of engineered particle parameter adjustments to meet / match desired cell subpopulation metrics are provided in Figure 4 Methods for adjusting the properties of engineered particles are described herein. Embodiments of how to adjust the forward scatter, side scatter, and surface properties of engineered particles are provided in Figure 5 and Figure 6 Provided in. The ability to adjust a series of parameters including engineered particle components and their concentrations allows the ability to adjust particles to simulate, for example, the characteristics of red blood cells. For example, in order to simulate the Coulter volume of red blood cells (which is related to impedance), the actual volume and / or porosity of the particles can be adjusted. It should be understood that the Coulter volume of the particles (as measured by the Coulter principle) depends on the amount of electrolytes replaced by the particles. For cells with complete membranes, their Coulter volumes are similar to their actual volumes. For the particles described herein, the amount of electrolytes replaced depends on the actual volume of the particles and the porosity of the hydrogel. This allows the Coulter volume of the particles of the present disclosure to be adjusted according to or independently of the actual size of the particles of the present disclosure. In the example of red blood cells, red blood cell simulation particles with diameters varying between 5 μm and 10 μm can be synthesized by adjusting conditions such as microfluidic pressure or microfluidic device design. As needed, the Coulter volume of these red blood cell simulation particles can be designed to be higher than, lower than or equal to the actual red blood cells or the conventional and / or abnormal subpopulations therein. Optical properties

[0038] As provided above, on the one hand, the present disclosure provides individually engineered particles, each of which has one or more properties substantially similar to one or more properties of a target cell (e.g., erythrocytes, reticulocytes, or platelets). In embodiments, the one or more properties may be optical properties. In embodiments, wherein the one or more properties include optical properties, the optical properties include side scattering characteristics, forward scattering characteristics, angled light scattering characteristics, or secondary marker characteristics, such as fluorescent marker characteristics, absorption characteristics, fluorescence characteristics, or emission characteristics.

[0039] In an embodiment, the optical properties are substantially similar to the absorption properties of red blood cells. Red blood cell absorption of light drops rapidly at about 600 nm and above, with slight variations if the red blood cells are bound to oxygen or not. The spectral waveform of hemoglobin is provided at omic.org / spectra / hemoglobin. Dyes can also be used to simulate the absorption of red blood cells.

[0040] In embodiments, the dye having an absorbance substantially similar to that of hemoglobin is a red dye. In embodiments, the dye having an absorbance substantially similar to that of hemoglobin has a peak excitation wavelength (nm) of about 600 to about 750, e.g., 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 28, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711 , 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749 or 750, including all ranges therein.

[0041] In embodiments, the dye having an absorbance substantially similar to that of hemoglobin is a red dye. In embodiments, the dye having an absorbance substantially similar to that of hemoglobin has a peak emission wavelength (nm) of about 625 to about 775, e.g., 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665 53, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736 , 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, or 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774 or 775, including all ranges therein.

[0042] In embodiments, the dye is Allura Red AC (also known as Red 40). In embodiments, the dye is India ink. In embodiments, the dye is EPolight 2717. In embodiments, the engineered particles are derivatized with one or more of the following fluorescent dyes: 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein; S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether-alanine-carboxamide or succinimidyl ester; 5-carboxyfluorescein succinimidyl ester; 6-carboxyfluorescein succinimidyl ester; Imidoester; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine 5-isothiocyanate; 6-(fluorescein-5-formylamino)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-formylamino)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon 488 Carboxylic acid or succinimide ester; Oregon 488 Isothiocyanate; Oregon 488-X Succinimidyl ester; Oregon 500 Carboxylic acid; Oregon 500 Carboxylic acid, succinimide ester or triethylammonium salt; Oregon 514 Carboxylic acid; Oregon 514 Carboxylic acid or succinimide ester; Rhodamine Green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green TM Carboxylic acid, trifluoroacetamide or succinimidyl ester; RhodamineGreen TM -X succinimidyl ester or hydrochloride; RhodolGreen TMCarboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfonyl rhodamine or bis(succinimidyl ester); 5-(and-6)carboxynaphthofluorescein, 5-(and-6)carboxynaphthofluorescein succinimidyl ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester; 6-carboxyrhodamine 6G succinimidyl ester; 5-(and-6)-carboxyrhodamine 6G succinimidyl ester; 5-carboxy-2',4',5',7'-tetrabromosulfonylfluorescein succinimidyl ester or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7 carboxylic acid or succinimide ester; Rhodamine RedTM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Sulfonyl; Texas Sulfonyl chloride; Texas STP ester or sodium salt; Texas Succinimide ester; Texas Succinimidyl ester; and X-rhodamine-5-(and -6)-isothiocyanate, commercially available from Invitrogen Dyes include but are not limited to FL, TMR STP ester, TR-X STP ester, 630 / 650-X STP ester, 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionic acid succinimide ester; 4,4-difluoro-4-bora-3a,4a-diaza-indacene-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-pentanoic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3 propionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a Diaza-indacene-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionyl)sulfoalanine, succinimide ester or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-indacene-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-indacene-3-propionic acid succinimide ester; 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-indacene-3-propionic acid; succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-indacene-3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1, 3-butadienyl)-4-bora-3a,4a-diaza-symmetric indacene-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-symmetric indacene-3-propionic acid succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-symmetric indacene-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-symmetric indacene-3-propionic acid; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-symmetric indacene-3-propionic acid; succinimide ester;4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-indacene-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-indacene-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-indacene-3-propionic acid succinimidyl ester; 6-(((4-( 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-indacen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimide ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-indacen-3-yl)phenylvinyloxy)acetyl)amino)hexanoic acid or succinimide ester; Alexa fluor dyes commercially available from Invitrogen, including but not limited to Alexa; 350 Carboxylic acid, Alexa 430 Carboxylic acid, Alexa 488 carboxylic acid, Alexa 532 Carboxylic acid, Alexa 546 Carboxylic acid, Alexa 555 Carboxylic acid; Alexa 568 carboxylic acid, Alexa 594 carboxylic acid, Alexa 633 carboxylic acid, Alexa 647 Carboxylic acid, Alexa 660 Carboxylic acid and Alexa 680 carboxylic acid; cyanine dyes commercially available from Amersham-Pharmacia Biotech, including but not limited to Cy3 NHS ester, Cy5 NHS ester, Cy5.5 NHS ester and Cy7 NHS ester.

[0043] In an embodiment, the engineered particles are "rainbow particles" containing a mixture of fluorophores (e.g., 4 fluorophores, 5 fluorophores, 6 fluorophores, seven fluorophores, or eight fluorophores). In this regard, the user selects the wavelength used to excite the particle based on the fluorophore being interrogated. Rainbow particles are commercially available from, for example, BD Biosciences (Catalog Nos. 556298 (mid-range FL1 fluorescence), 556286 (6 colors, 3.0-3.4 μm), 556288 (6 colors, 6.0-6.4 μm), 559123 (8 colors)) and from Spherotech in various diameters (e.g., Catalog Nos. RCP20-5 (4 colors), RCP-30-5 (6 peaks), RCP-30-5A (8 peaks)).

[0044] Hemoglobin-like molecules can be used to simulate the absorption of red blood cells. For example, heme is the precursor of hemoglobin. Hemoglobin can also come from humans, mice, cattle, sheep, birds, dogs, cats, pigs or plant sources. Legume hemoglobin can be derived from soybean plants and can be produced by bioengineered yeast. Hemoglobin variants include: Gower 1, Gower 2, hemoglobin Portland 1, hemoglobin Portland II, hemoglobin F, hemoglobin A, hemoglobin A2, hemoglobin F, hemoglobin D-Pubjab, hemoglobin H, hemoglobin Barts, hemoglobin S, hemoglobin C, hemoglobin E, hemoglobin AS, hemoglobin SC and hemoglobin Hopkins-2. Hemoglobin-like molecules include myoglobin, hemocyanin, earthworm hemoglobin, hemoglobin, vanabins, invertebrate hemoglobin, prairie globulin, leghemoglobin and cobalt globulin.

[0045] One or more surfaces of the particle can be functionalized, for example, to simulate one or more optical properties of the target cell or the marked target cell. Functionalized hydrogel particles can also include embedded beads or substances (such as biomolecules), as described above. In an embodiment, one or more hydrogel particles are functionalized by one or more fluorescent dyes, one or more cell surface markers (or their epitope binding regions) or a combination thereof. In an embodiment, hydrogel particles are formed by polymerizing at least one bifunctional monomer, and after formation, the hydrogel particles include one or more functional groups that can be used to further attach cell surface markers, epitope binding regions of cell surface markers, fluorescent dyes or a combination thereof. In an embodiment, the free functional group is an amine, a carboxyl, a hydroxyl or a combination thereof. Depending on the desired functionalization, it should be understood that a variety of bifunctional monomers can be used, for example, different chemicals and different molecules can be used to functionalize particles.

[0046] The engineered particles can be functionalized with any fluorescent dye known in the art, including The Fluorescent dyes listed in Handbook-A Guide to Fluorescent Probes and Labeling Technologies (incorporated herein by reference in its entirety for all purposes). Functionalization can be mediated by compounds containing free amine groups (e.g., allylamine) that can be incorporated into difunctional monomers used to form hydrogels, as discussed above.

[0047] Non-limiting examples of known fluorescent dyes that can be used to functionalize the surface of the particles described herein include: 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein; S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether, -alanine-carboxamide or succinimidyl ester; 5-carboxyfluorescein succinimidyl ester; 6-carboxyfluorescein succinimidyl ester; Imidyl esters; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-formamido)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-formamido)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon 488 Carboxylic acid or succinimide ester; Oregon 488 Isothiocyanate; Oregon 488-X Succinimidyl ester; Oregon 500 Carboxylic acid; Oregon 500 Carboxylic acid, succinimide ester or triethylammonium salt; Oregon 514 Carboxylic acid; Oregon 514 Carboxylic acid or succinimide ester; Rhodamine Green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green TM Carboxylic acid, trifluoroacetamide, or succinimidyl ester; Rhodamine Green TM -X succinimidyl ester or hydrochloride; RhodolGreen TMCarboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine or bis(succinimidyl ester); 5-(and-6)-carboxynaphthofluorescein; 5-(and-6)-carboxynaphthofluorescein succinimidyl ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G hydrochloride; 5-carboxyrhodamine 6G succinimidyl ester; 6-carboxyrhodamine 6G succinimidyl ester; 5-(and-6)-carboxyrhodamine 6G succinimidyl ester; 5-carboxy-2',4',5',7'-tetrabromosulfonefluorescein succinimidyl ester or bis-(diisopropylethylammonium) salt ; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7 carboxylic acid or succinimide ester; Rhodamine RedTM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Sulfonyl; Texas Sulfonyl chloride; Texas STP ester or sodium salt; Texas Succinimide ester; Texas succinimidyl esters; and X-rhodamine-5-(and -6)-isothiocyanate.

[0048] Other examples of fluorescent dyes for use with the particles described herein include, but are not limited to, those commercially available from Invitrogen. Dyes, including but not limited to FL, TMR STP ester, TR-X STP ester, 630 / 650-X STP ester, 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionic acid succinimide ester; 4,4-difluoro-4-bora-3a,4a-diaza-indacene-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-pentanoic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a -diaza-indacene-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionyl)sulfoalanine, succinimide ester or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-indacene-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-indacene-3-propionic acid succinimide ester; 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-indacene-3-propionic acid; succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-indacene-3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1 ,3-butadienyl)-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-sym-indacene-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid; succinimide ester;4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-indacene-8-propionic acid; 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-indacene-8-propionic acid succinimidyl ester; 4,4-Difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-indacene-3-propionic acid succinimidyl ester ; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-indacen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimide ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-indacen-3-yl)phenylvinyloxy)acetyl)amino)hexanoic acid or succinimide ester. ;

[0049] In embodiments, fluorescent dyes used to derivatize the surface of one or more particles include, but are not limited to, Alexa fluorescent dyes commercially available from Invitrogen, including, but not limited to, Alexa 350 Carboxylic acid, Alexa 430 Carboxylic acid, Alexa 488 carboxylic acid, Alexa 532 Carboxylic acid, Alexa 546 Carboxylic acid, Alexa 555 carboxylic acid, Alexa 568 carboxylic acid, Alexa 594 carboxylic acid, Alexa 633 carboxylic acid, Alexa 647 Carboxylic acid, Alexa 660 carboxylic acid and Alexa 680 carboxylic acid. In embodiments, fluorescent dyes for use with the hydrogel particles and methods described herein include cyanine dyes commercially available from Amersham-Pharmacia Biotech, including but not limited to Cy3 NHS ester, Cy5 NHS ester, Cy5.5 NHS ester, and Cy7 NHS ester.

[0050] It is within the ordinary skill in the art to select the appropriate dye or dyes based on the desired absorption or spectral excitation and emission characteristics of the particle.

[0051] In embodiments, multiple particles are used to determine the dynamic range and / or sensitivity of the detection of a specific cell surface marker or its combination on a target cell population. For example, a hydrogel particle population can be adjusted to have the SSC and / or FSC characteristics of a target cell, and a subpopulation of hydrogel particles is derivatized with a specific number of copies of a cell surface marker (e.g., a cell surface receptor or its domain, such as its epitope binding region). For example, a separate subpopulation of hydrogel particles can each be derivatized to have a unique number of copies, for example, a subpopulation will contain 100 copies of a cell surface marker, a second subpopulation will contain 1,000 copies of the same cell surface marker, and a third subpopulation will contain 10,000 copies of the same cell surface marker, etc. For the corresponding cell surface marker, the hydrogel particle population is fluorescently stained, and the fluorescence of the hydrogel particles in each subpopulation is detected. In this regard, a subpopulation of hydrogel particles can be used to generate a fluorescence emission standard curve of a target cell with a corresponding cell marker. The cell surface marker can be any cell surface marker or its binding region provided or a cell surface marker known to a person of ordinary skill in the art.

[0052] As used herein, "substantially similar" means at least 40% similar, at least 50% similar, at least 60% similar, at least 70% similar, at least 80% similar, at least 90% similar, at least 95% similar, at least 96% similar, at least 97% similar, at least 98% similar, or at least 99% similar.

[0053] In embodiments, the disclosed hydrogel particles have a refractive index (RI) greater than about 1.10, greater than about 1.15, greater than about 1.20, greater than about 1.25, greater than about 1.30, greater than about 1.35, greater than about 1.40, greater than about 1.45, greater than about 1.50, greater than about 1.55, greater than about 1.60, greater than about 1.65, greater than about 1.70, greater than about 1.75, greater than about 1.80, greater than about 1.85, greater than about 1.90, greater than about 1.95, greater than about 2.00, greater than about 2.10, greater than about 2.20, greater than about 2.30, greater than about 2.40, greater than about 2.50, greater than about 2.60, greater than about 2.70, greater than about 2.80, or greater than about 2.90.

[0054] In embodiments, the disclosed hydrogel particles have a refractive index (RI) of about 1.10 to about 3.0, or about 1.15 to about 3.0, or about 1.20 to about 3.0, or about 1.25 to about 3.0, or about 1.30 to about 3.0, or about 1.35 to about 3.0, or about 1.4 to about 3.0, or about 1.45 to about 3.0, or about 1.50 to about 3.0, or about 1.6 to about 3.0, or about 1.7 to about 3.0, or about 1.8 to about 3.0, or about 1.9 to about 3.0, or about 2.0 to about 3.0.

[0055] In embodiments, the disclosed hydrogel particles have a refractive index (RI) of less than about 1.10, less than about 1.15, less than about 1.20, less than about 1.25, less than about 1.30, less than about 1.35, less than about 1.40, less than about 1.45, less than about 1.50, less than about 1.55, less than about 1.60, less than about 1.65, less than about 1.70, less than about 1.75, less than about 1.80, less than about 1.85, less than about 1.90, less than about 1.95, less than about 2.00, less than about 2.10, less than about 2.20, less than about 2.30, less than about 2.40, less than about 2.50, less than about 2.60, less than about 2.70, less than about 2.80, or less than about 2.90.

[0056] The SSC of the disclosed hydrogel particles is most meaningfully measured compared to the target cells. In embodiments, the SSC of the disclosed hydrogel particles is within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% of the SSC of the target cells, as measured by a hematology analyzer.

[0057] In embodiments, the SSC of the hydrogel particles is adjusted by incorporating a high refractive index molecule (or a plurality thereof) into the hydrogel. In embodiments, the high refractive index molecule is provided in the hydrogel particles, and in further embodiments, the high refractive index molecule is colloidal silica, an alkyl acrylate, an alkyl methacrylate, or a combination thereof. Thus, in embodiments, the hydrogel particles of the present disclosure comprise an alkyl acrylate and / or an alkyl methacrylate. In embodiments, the concentration of the monomer is adjusted to further adjust the refractive index of the hydrogel particles.

[0058] The alkyl acrylate or alkyl methacrylate may contain 1 to 18, 1 to 8 or 2 to 8 carbon atoms in the alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, 2-ethylhexyl, heptyl or octyl. The alkyl group may be branched or straight chain.

[0059] In embodiments, hydrogel particles of the present disclosure have material modulus properties (eg, elasticity) that more closely resemble those of target cells compared to polystyrene beads having the same diameter.

[0060] In an embodiment, a hemoglobin molecule or a dye molecule is covalently attached to a cleavable hydrogel. Non-limiting examples of covalent linkers include disulfide linkers, ester linkers, amine linkers, thiol linkers, and carbonyl linkers. In an embodiment, a hemoglobin molecule or a dye molecule is non-covalently attached to a cleavable hydrogel. Non-limiting examples of non-covalent linkers include streptavidin-biotin, neutravidin-biotin, and affinity tags (such as His-tags, GST-tags, Halo-tags, SNAP-tags). Shape / Size

[0061] In embodiments, the engineered particle (e.g., hydrogel) has the morphology or shape of a target cell. Mammalian erythrocytes are typically shaped as biconcave disks: flattened and concave in the center, with a dumbbell-shaped cross-section, and with a donut-like rim on the edge of the disk. Erythrocytes may have a diameter of 4.84*volume. 2 / 3 The dimensionless "sphericity index" of the area is a characteristic shape. "Distribution of Size and Shape in Populations of Normal Human Red Cells," Canham and Burton, Circulation Research, Vol. XXII, March 1968, which is incorporated by reference in its entirety for all purposes.

[0062] There are some shape-related exceptions in the Artiodactyls (even-toed ungulates, including cattle, deer, and their close relatives), which display a variety of red blood cell morphologies: small, highly ovoid cells in llamas and camels (Camelidae), tiny spherical cells in mouse deer (Tragulidae), and spindle-, lanceolate, crescent-, and irregular polygonal and other angular cells in red deer and elk (Cervidae).

[0063] In embodiments, the engineered particles (eg, hydrogels) are flexible and deformable to squeeze through microscopic capillaries.

[0064] Droplet size is related to microfluidic channel size. Microfluidic channels can have any size, for example having a maximum dimension perpendicular to fluid flow of less than about 5 mm or 2 mm, or less than about 1 mm, or less than about 500 μm, less than about 200 μm, less than about 100 μm, less than about 60 μm, less than about 50 μm, less than about 40 μm, less than about 30 μm, less than about 25 μm, less than about 10 μm, less than about 3 μm, less than about 1 μm, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm.

[0065] The droplet size can be adjusted by adjusting the relative flow rates. In embodiments, the droplet diameter is equal to the width of the channel, or within about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the channel width.

[0066] The hydrogel particles of the present disclosure are substantially similar in size to the droplets of which they are composed. Thus, in embodiments, the hydrogel particles have a diameter of less than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800 μm or less than 1000 μm in diameter. In embodiments, the hydrogel particles have a diameter greater than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800 μm, or greater than 1000 μm in diameter.

[0067] In embodiments, the size (e.g., diameter, width, thickness) of the hydrogel particles of the present disclosure is substantially similar to the target cells they are modeled in. Thus, in embodiments, the hydrogel particles have a diameter of less than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800 μm or less than 1000 μm in diameter. In an embodiment, the hydrogel particles have a diameter greater than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800 μm, or greater than 1000 μm. In an embodiment, the hydrogel particles have a diameter in the range of 2.5 μm to 100 μm. In an embodiment, the hydrogel particles have a diameter between about 2.5 μm and about 25 μm, between about 3 μm and about 20 μm, between about 3.5 μm and about 15 μm, between about 4 μm and about 12 μm, between about 5 μm and about 10 μm, between about 6 μm and about 9 μm, or between about 7 μm and about 8 μm. In addition, in embodiments, the hydrogel particles have a width of less than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800 μm or less than 1000 μm. In embodiments, the hydrogel particles have a width of greater than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800 μm or greater than 1000 μm. In an embodiment, the hydrogel particles have a width in the range of 2.5 μm to 100 μm. In an embodiment, the hydrogel particles have a width between about 2.5 μm and about 25 μm, between about 3 μm and about 20 μm, between about 3.5 μm and about 15 μm, between about 4 μm and about 12 μm, between about 5 μm and about 10 μm, between about 6 μm and about 9 μm, or between about 7 μm and about 8 μm.In addition, in embodiments, the hydrogel particles have a thickness of less than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800 μm or less than 1000 μm. In embodiments, the hydrogel particles have a thickness of greater than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800 μm or greater than 1000 μm. In an embodiment, the hydrogel particles have a thickness in the range of 2.5 μm to 100 μm. In an embodiment, the hydrogel particles have a thickness between about 2.5 μm and about 25 μm, between about 3 μm and about 20 μm, between about 3.5 μm and about 15 μm, between about 4 μm and about 12 μm, between about 5 μm and about 10 μm, between about 6 μm and about 9 μm, or between about 7 μm and about 8 μm.

[0068] In an embodiment, each engineered particle (e.g., a hydrogel) is substantially similar to the diameter or volume of a red blood cell, which results in the engineered particle having an impedance substantially similar to that of a human red blood cell. Red blood cells typically have a diameter of 7-9 um. Red blood cells typically have a volume of 80-140 femtoliters. "Distribution of Size and Shape in Populations of Normal Human Red Cells," Canham and Burton, Circulation Research, Vol. XXII, March 1968, which is incorporated by reference in its entirety for all purposes.

[0069] In embodiments, the red blood cell control composition comprises one or more hydrogel particle populations, wherein the average diameter of at least one hydrogel particle population ranges from about 1 μm to about 20 μm (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 μm). In embodiments, the average diameter of at least one hydrogel particle population ranges from about 1 μm to about 15 μm. In embodiments, the average diameter of at least one hydrogel particle population ranges from about 2 μm to about 15 μm. In embodiments, the average diameter of at least one hydrogel particle population ranges from about 5 μm to about 15 μm. In embodiments, the average diameter of at least one hydrogel particle population ranges from about 5 μm to about 10 μm. In embodiments, the average diameter of at least one hydrogel particle population ranges from about 7 μm to about 9 μm.

[0070] In embodiments, the hydrogel particles of the present disclosure are spherical in shape. In embodiments, the hydrogel particles of the present disclosure are at least one of concave and biconcave in shape.

[0071] In embodiments, the volume of the hydrogel particles of the present disclosure is substantially similar to the target cells they are modeled for. For example, when the target cells are red blood cells, the hydrogel particles can have a volume of more than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800 fL or greater than 1000 fL. In embodiments, the hydrogel particles have a volume in the range of 50 fL to 200 fL. In an embodiment, the hydrogel particles have a volume of between about 75fL and about 175fL, between about 100fL and about 150fL, between about 110fL and about 140fL, between about 120fL and about 130fL, between about 122fL and about 128fL, between about 123fL and about 127fL, or between about 124fL and about 126fL. In an embodiment, the hydrogel particles have a volume between 75fL and 175fL. In an embodiment, the hydrogel particles have a volume between about 80fL and about 100fL. In an embodiment, the hydrogel particles have a volume between about 85fL and about 95fL. In an embodiment, the hydrogel particles have a volume between about 130fL and about 170fL. In an embodiment, the hydrogel particles have a volume between about 140fL and about 160fL. In embodiments, the hydrogel particles have a volume between about 145 fL and about 155 fL.

[0072] In embodiments, the hydrogel particles of the present disclosure do not comprise agarose.

[0073] In an embodiment, the hydrogel particles are supported by suspension polymerization, which is also known in the art as beading, beaded or granulation polymerization (see Elbert (2011). Acta Biomater. 7, pp. 31-56, which is incorporated herein by reference in its entirety for all purposes. In suspension polymerization, the monomer is insoluble in a continuous phase, such as an aqueous solution of monomer in a continuous oil phase. In suspension polymerization, polymerization initiation occurs within the monomer-rich droplets, and there is more than one free radical per droplet at any time. In an embodiment, the monomer phase comprises a monomer that can be a difunctional monomer or multiple monomer species (comonomers, which can be multiple difunctional monomers). In an embodiment, the monomer phase comprises an initiator and / or a crosslinker.

[0074] Emulsion polymerization can also be used to form the hydrogel particles described herein. In emulsion polymerization, the monomer is poorly cleaved in the continuous phase, similar to suspension polymerization, however, polymerization initiation occurs outside the monomer droplets (see Elbert (2011). Acta Biomater. 7, pp. 31-56, which is incorporated herein by reference in its entirety for all purposes). In an emulsion polymerization embodiment, if a surfactant is present, the initiator causes chain growth of the monomer (or comonomer) dissolved in the continuous phase or the monomer contained in the micelle.

[0075] In an embodiment, hydrogel particles are formed by precipitation polymerization, for example as described in Elbert (2011). Acta Biomater. 7, pp. 31-56, which is incorporated herein by reference in its entirety for all purposes. Precipitation polymerization is a technique for producing microparticles using differences in the solubility of monomers and polymers. Specifically, it is known that larger polymer chains generally have lower solubility than smaller polymer chains. Therefore, above a specific molecular weight, phase separation can be advantageous. Precipitation polymerization initially begins with solution polymerization in a single-phase, homogeneous system. In an embodiment, shortly after the start of polymerization, there is a relatively high concentration of polymer chains, which is conducive to phase separation by nucleation. As the polymerization proceeds, the concentration of polymer chains is low, and existing particles capture chains before new particles nucleate. Therefore, nucleation of particles occurs only in a short period of time shortly after the start of the reaction, which results in a narrow particle size distribution in an embodiment. Additional methods include, but are not limited to, photolithographic particle formation (Helgeson et al. (2011). Curr. Opin. Colloid. Interface Sci. 16, pp. 106-117, which is incorporated herein by reference in its entirety for all purposes), membrane emulsification (e.g., by the microsieve emulsification technique described by Nanomi BV (The Netherlands)) and microchannel emulsification (Sugiura et al. (2002). Languimir 18, pp. 5708-5712, which is incorporated herein by reference in its entirety), and batch emulsification (SNF Floerger, available at snf.com.au / downloads / Emulsion_Handbook_E.pdf, which is incorporated herein by reference in its entirety).

[0076] In embodiments, hydrogel particles are formed in a microfluidic device having two oil channels that are centered on a central stream of an aqueous monomer solution. In this embodiment, droplets are formed at the interface of the two channels and the central stream to break the droplets apart in an oil-in-water emulsion. After the droplets are formed, in embodiments, they are stabilized prior to polymerization, for example by adding a surfactant to the oil phase. However, in embodiments, the droplets are not stabilized prior to polymerization. In embodiments, polymerization of the monomer is triggered by adding a promoter (e.g., N,N,N',N'tetramethylethylenediamine) to one or both oil channels after the initial droplets are formed.

[0077] The aqueous monomer solution as provided above may comprise a single monomer species or a plurality of monomer species. The aqueous monomer solution may comprise a comonomer, a difunctional monomer, or a combination thereof. In an embodiment, the monomer or monomers may comprise a difunctional monomer, such as one of the monomers described above. As described below, the comonomer may be used, for example, to adjust the forward scattering or the side scattering by adjusting the refractive index of the hydrogel particles.

[0078] In embodiments, the center stream of the aqueous monomer solution comprises a crosslinking agent, such as N,N'-bisacrylamide. In further embodiments, the center stream of the aqueous monomer solution comprises a crosslinking agent and a accelerator in addition to the monomers. In yet further embodiments, the aqueous monomer solution comprises an initiator, such as an oxidizing agent, such as ammonium persulfate.

[0079] Forward scattering is adjusted by adjusting the refractive index of the gel by adding comonomers allyl acrylate and allyl methacrylate. Forward scattering can also be adjusted with side scattering nanoparticles containing sufficient optical resolution / size / density, including but not limited to higher density colloidal suspensions of silica and / or PMMA particles. Side scattering of the droplets is adjusted by adding a colloidal suspension of silica nanoparticles and / or PMMA (poly(methyl methacrylate)) particles (about 100 nm) to the central aqueous phase before polymerization.

[0080] Engineered particles (e.g., hydrogels) can be made and adjusted to adjust their electrical properties. Electrical properties can include, for example, capacitance, impedance, etc. Engineered particles (e.g., hydrogels) can be made for calibration of hematology analyzers, such as for calibration of Coulter counters. In an embodiment, the capacitance of the particles is adjusted by changing the amount of hydrogel monomers in the composition. For example, polyaniline, polyacetylene; polystyrene; polypyrrole (X=NH) and polythiophene (X=S) comonomers; and polyaniline (X=NH / N) and polyphenylene sulfide (X=S) comonomer concentrations can all be adjusted to change the capacitance. In an embodiment, the concentration of one or more of these monomers is increased to increase the capacitance of the hydrogel particles. In an embodiment, the diameter or volume of the engineered particles (e.g., hydrogels) can be adjusted to simulate the impedance or capacitance of red blood cells, reticulocytes, or platelets. Red blood cell control

[0081] The red blood cell control samples described herein can be adjusted to correspond to normal blood samples of healthy people or disease states. This can be done by adjusting the following amounts and / or ratios: (i) one or more cleavable hydrogel particle populations having an impedance substantially similar to the impedance of human red blood cells having an average diameter; and (ii) a population of hemoglobin molecules or a population of dye molecules having an absorbance substantially similar to that of hemoglobin. For example, Table 1 below describes the following properties of normal (healthy) blood samples as measured by a hematology analyzer: red blood cell count (RBC), hemoglobin concentration (HBG), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red blood cell distribution width (RDW), and nucleated red blood cells (NRBC). As described in more detail below, the red blood cell control compositions of the present disclosure can be adjusted to match the properties in Table 1 and correspond to normal (healthy) samples, or the control compositions can be adjusted to correspond to various disease states by having properties outside the range shown in Table 1. For example, in embodiments, the number, volume, and distribution of the same hydrogel particles can be modified to correspond to target RBC, MCV, and / or RDW, respectively. Similarly, in embodiments, the concentration of hemoglobin or dye having substantially similar absorbance can be modified to correspond to target HGB and / or MCV. Table 1. Characteristics of normal (healthy) red blood cell samples Male - Low Male - High Female - Low Female - High <![CDATA[RBC(1x 10 9 / mL)]]> 4.2 5.9 4.2 5.9 HGB(g / dL) 14 17 12 16 MCV(fL) 80.0 100.0 80.0 100.0 MCH(pg) 28 32 28 32 RDW(%) 11 16 11 16 NRBC >100 / 100 μL >100 / 100 μL (i) Normal red blood cell characteristics

[0082] In an embodiment, the RBCs (1 x 10 9 / mL) ranged from about 4.2 to about 5.91x10 9 (e.g., about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, or about 5.91 x 10 9 , including all values ​​and ranges therein).

[0083] In embodiments, the range of HGB (g / dL) of the red blood cell control composition is about 12 to about 17 g / dL (e.g., about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, or about 17 g / dL, including all values ​​and ranges therein). In embodiments, the range of HGB (g / dL) of the red blood cell control composition for males is about 14 to about 17 g / dL (e.g., about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, or about 17 g / dL). In embodiments, the HGB (g / dL) of the red blood cell control composition for a female ranges from about 12 to about 16 g / dL (e.g., about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, or about 16 g / dL).

[0084] In embodiments, the MCV (fL) of the red blood cell control composition ranges from about 80 to about 100 fL (e.g., about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 fL, including all values ​​and ranges therein).

[0085] In embodiments, the MCH (pg) of the red blood cell control composition ranges from about 28 to about 32 fL (e.g., about 28, about 28.5, about 29, about 29.5, about 30, about 30.5, about 31, about 31.5, or about 32 pg, including all values ​​and ranges therein).

[0086] In embodiments, the RDW (%) of the red blood cell control composition ranges from about 11% to about 16% (e.g., about 11%, about 11.5%, about 12%, about 12.5%, about 13, about 13.5%, about 14%, about 14.5%, about 15%, about 15.5%, or about 16%, including all values ​​and ranges therein).

[0087] In embodiments, the red blood cell control composition has less than about 100 / 100 μL of NRBCs, e.g., less than or equal to about 99.5, about 99, about 98, about 97, about 96, about 95, about 94, about 93, about 92, about 91, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 50, about 45, about 40, about 35, about 30, about 25, about 20 / 100 μL, including all values ​​and subranges therein. In embodiments, the NRBCs of the red blood cell control composition range from 99.5 to 20 / 100 μL, e.g., about 99.5, about 99, about 98, about 97, about 96, about 95, about 94, about 93, about 92, about 91, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 50, about 45, about 40, about 35, about 30, about 25, about 20 / 100 μL, including all values ​​and subranges therein. (ii) Disease

[0088] As discussed herein, control samples for disease states can be prepared by adjusting the amount of red blood cell control.

[0089] In an embodiment, the composition has less than about 4.2 1 x 10 RBCs. 9 / mL, for example, less than or equal to about 4, about 3.75, about 3.5, about 3.25, about 3, about 2.75, about 2.5, about 2.25, about 2, about 1.75, about 1.5, about 1.25, about 1, about 0.75, about 0.5, or about 0.25 1 x 10 9 / mL, including all values ​​and ranges therein. In embodiments, the range of RBCs of the composition is about 1-4 1x 10 9 / mL, for example, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.25, about 2.5, about 2.75, about 3, about 3.25, about 3.5, about 3.75, or about 4 1 x 10 9 / mL, including all values ​​and ranges therein. In embodiments, the composition has RBCs greater than about 5.9 1 x 10 9 / mL, for example, greater than or equal to about 6, about 6.25, about 6.5, about 6.75, about 7, about 7.25, about 7.5, about 7.75, about 8, about 8.25, about 8.5, about 8.75, about 9, about 9.25, about 9.5, about 9.75, or about 10 1x 10 9 / mL, including all values ​​and ranges therein. In embodiments, the composition has a range of RBCs from about 6 to about 10 1x 10 9 / mL, for example, about 6, about 6.25, about 6.5, about 6.75, about 7, about 7.25, about 7.5, about 7.75, about 8, about 8.25, about 8.5, about 8.75, about 9, about 9.25, about 9.5, about 9.75, about 10 1x 10 9 / mL, including all values ​​and ranges therein.

[0090] In embodiments, the HGB (g / dL) of the red blood cell control composition (e.g., for women) is less than about 12 g / dL, e.g., less than or equal to about 11.75, about 11.5, about 11.25, about 11, about 10.75, about 10.5, about 10.25, about 10, about 9.75, about 9.5, about 9.25, about 9, about 8.75, about 8.5, about 8.25, about 8, about 7.75, about 7.5, about 7.25, about 7, about 6.75, about 6.5, about 6.25, about 6, about 5.75, about 5.5, about 5.25, about 5, about 4.75, about 4.5, about 4.25, about 4 g / dL. In embodiments, the HGB (g / dL) of a red blood cell control composition (e.g., for women) ranges from about 11.5 to about 6.5 g / dL, e.g., about 11.5, about 11.25, about 10, about 10.75, about 10.5, about 10.25, about 9, about 9.75, about 9.5, about 9.25, about 8, about 8.75, about 8.5, about 8.25, about 7, about 7.75, about 7.5, about 7.25, about 6, about 6.75, or about 6.5 g / dL.

[0091] In embodiments, the HGB (g / dL) of the red blood cell control composition (e.g., for males) is less than about 14 g / dL, e.g., less than or equal to about 13.75, about 13.5, about 13.25, about 13, about 12.75, about 12.5, about 12.25, about 12, about 11.75, about 11.5, about 11.25, about 11, about 10.75, about 10.5, about 10.2 5, about 10, about 9.75, about 9.5, about 9.25, about 9, about 8.75, about 8.5, about 8.25, about 8, about 7.75, about 7.5, about 7.25, about 7, about 6.75, about 6.5, about 6.25, about 6, about 5.75, about 5.5, about 5.25, about 5, about 4.75, about 4.5, about 4.25, about 4 g / dL, including all values ​​and subranges therein. In embodiments, the HGB (g / dL) of the red blood cell control composition (e.g., for males) ranges from 13.5 to about 7.5 g / dL, e.g., about 13.5, about 13.25, about 13, about 12.75, about 12.5, about 12.25, about 12, about 11.75, about 11.5, about 11.25, about 10, about 10.75, about 10.5, about 10.25, about 9, about 9.75, about 9.5, about 9.25, about 8, about 8.75, about 8.5, about 8.25, about 7, about 7.75, or about 7.5 g / dL, including all values ​​and subranges therein.

[0092] In embodiments, the HGB (g / dL) of the red blood cell control composition (e.g., for males) is greater than about 17 g / dL, e.g., about 17.25, about 17.5, about 17.75, about 18, about 18.25, about 18.5, about 18.75, about 19, about 19.25, about 19.5, about 19.75, about 20, about 20.25, about 20.5, about 20.75, about 21, about 21.25, about 21.5, about 21.75, about 22, about 23.25, about 23.5, about 23.75, about 24, about 24.25, about 24.5, about 24.75, about 25, about 25.25, about 25.5, about 25.75, or about 26 g / dL, including all values ​​and ranges therein. In embodiments, the HGB (g / dL) of the red blood cell control composition (e.g., for males) ranges from about 17.5 g / dL to about 22.5 g / dL, e.g., about 17.5, about 17.75, about 18, about 18.25, about 18.5, about 18.75, about 19, about 19.25, about 19.5, about 19.75, about 20, about 20.25, about 20.5, about 20.75, about 21, about 21.25, about 21.5, about 21.75, or about 26 g / dL, including all values ​​and subranges therein.

[0093] In embodiments, the HGB (g / dL) of the red blood cell control composition (e.g., for women) is greater than about 16 g / dL, e.g., about 16.25, about 16.5, about 16.75, about 17, about 17.25, about 17.5, about 17.75, about 18, about 18.25, about 18.5, about 18.75, about 19, about 19.25, about 19.5, about 19.75, about 19. About 20, about 20.25, about 20.5, about 20.75, about 21, about 21.25, about 21.5, about 21.75, about 22, about 23.25, about 23.5, about 23.75, about 24, about 24.25, about 24.5, about 24.75, about 25, about 25.25, about 25.5, about 25.75, or about 26 g / dL, including all values ​​and ranges therein. In embodiments, the HGB (g / dL) of the red blood cell control composition (e.g., for females) ranges from about 16.5 g / dL to about 21.5 g / dL, e.g., about 16.5, about 16.75, about 17, about 17.25, about 17.5, about 17.75, about 18, about 18.25, about 18.5, about 18.75, about 19, about 19.25, about 19.5, about 19.75, about 20, about 20.25, about 20.5, about 20.75, about 21, about 21.25, or about 21.5 g / dL, including all values ​​and subranges therein.

[0094] In embodiments, the MCV (fL) of the red blood cell control composition is less than 80 fL, e.g., less than or equal to about 79.5, about 79, about 78, about 77, about 76, about 75, about 74, about 73, about 72, about 71, about 70, about 65, about 60, about 50, about 45, about 40, about 35, about 30, about 25, about 20 fL, including all values ​​and subranges therein. In embodiments, the MCV (fL) of the red blood cell control composition ranges from about 79.5 to about 20 g / dL, e.g., about 79.5, about 79, about 78, about 77, about 76, about 75, about 74, about 73, about 72, about 71, about 70, about 65, about 60, about 50, about 45, about 40, about 35, about 30, about 25, about 20 fL, including all values ​​and subranges therein. In embodiments, the MCV (fL) of the red blood cell control composition is greater than 100 fL, e.g., greater than or equal to about 100.5, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200 fL, including all values ​​and subranges therein. In embodiments, the MCV (fL) of the red blood cell control composition ranges from about 100.5 to about 200 g / dL, e.g., about 100.5, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200 fL, including all values ​​and subranges therein.

[0095] In embodiments, the MCH (pg) of the red blood cell control composition is less than 28 pg, e.g., less than or equal to about 27.5, about 27, about 26, about 25, about 24, about 23, about 22, about 21, about 20, about 18, about 16, about 14, about 12, about 10 pg, including all values ​​and subranges therein. In embodiments, the MCH (pg) of the red blood cell control composition ranges from about 27.5 to about 10 pg, e.g., about 27.5, about 27, about 26, about 25, about 24, about 23, about 22, about 21, about 20, about 18, about 16, about 14, about 12, about 10 pg, including all values ​​and subranges therein. In embodiments, the MCH (pg) of the red blood cell control composition is greater than 32 pg, e.g., greater than or equal to about 32.5, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 42, about 46, about 48, about 50, about 52, about 54 pg, including all values ​​and subranges therein. In embodiments, the MCH (pg) of the red blood cell control composition ranges from about 32.5 to about 54 pg, e.g., about 32.5, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 42, about 46, about 48, about 50, about 52, about 54 pg, including all values ​​and subranges therein.

[0096] In embodiments, the RDW of the red blood cell control composition is less than 11%, for example, less than or equal to about 10.5%, about 10%, about 9.5%, about 9%, about 8.5%, about 8%, about 7.5%, about 7%, about 6.5%, about 6%, about 5.5%, about 5%, including all values ​​and subranges therein. In embodiments, the RDW of the red blood cell control composition ranges from about 10.5% to about 5%, for example, about 10.5%, 10%, about 9.5%, about 9%, about 8.5%, about 8%, about 7.5%, about 7%, about 6.5%, about 6%, about 5.5%, about 5%, including all values ​​and subranges therein. In embodiments, the RDW of the red blood cell control composition is greater than 16%, e.g., greater than or equal to about 16.5%, about 17%, about 17.5%, about 18%, about 18.5%, about 19%, about 19.5%, about 20%, about 20.5%, about 21%, about 21.5%, about 22%, including all values ​​and subranges therein. In embodiments, the RDW of the red blood cell control composition ranges from about 16.5% to about 22%, e.g., about 16.5%, about 17%, about 17.5%, about 18%, about 18.5%, about 19%, about 19.5%, about 20%, about 20.5%, about 21%, about 21.5%, about 22%, including all values ​​and subranges therein.

[0097] In embodiments, the NRBCs of the red blood cell control composition are greater than about 100 / 100 μL, e.g., greater than or equal to about 100.5, about 101, about 102, about 013, about 104, about 015, about 106, about 107, about 108, about 109, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150 / 100 μL, including all values ​​and subranges therein. In an embodiment, the range of NRBCs in the red blood cell control composition is about 100.5 to about 150 / 100 μL, for example, about 100.5, about 101, about 102, about 013, about 104, about 015, about 106, about 107, about 108, about 109, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150 / 100 μL, including all values ​​and sub-ranges therein.

[0098] In embodiments, the properties of the control red blood cell composition are modified to produce a positive control for hematological detection of a disorder or disease. In embodiments, the disorder or disease is NRBC elevation, bone marrow failure, anemia, red blood cell enzyme deficiency, red blood cell membrane disorder, hemoglobinopathy, polycythemia, hemochromatosis, hyperhemoglobin, hypohemoglobin, polycythemia, anisocytosis, macrocytosis, microcytosis or leukemia. For example, in embodiments, the average size of the hydrogel will be reduced to outside the detection range of healthy adults to produce a hematological reading simulating the hematological reading of a patient with microcytosis. In embodiments, the average size of the hydrogel will be increased to outside the detection range of healthy adults to produce a hematological reading simulating the hematological reading of a patient with macrocytosis. In embodiments, the size dispersion (RDW) of the hydrogel is increased to outside the range of healthy adults to simulate the hematological reading of a patient with nutritional deficiency or anisocytosis. In embodiments, the concentration of the hydrogel is increased to simulate polycythemia. An exemplary control red blood cell composition for simulating a disease is shown in Fig. 8A and Figure 8B middle.

[0099] In embodiments, the concentration of hemoglobin (human or another animal, such as cattle) or dye in the composition is reduced with or without changing the concentration of the hydrogel to simulate the hematological readings of a patient with iron deficiency anemia or too little hemoglobin. In embodiments, the concentration of hemoglobin (human or another animal, such as cattle) or dye in the composition is increased with or without changing the concentration of the hydrogel to simulate the hematological readings of a patient with too much hemoglobin. Exemplary HGB mimetics are shown in Figure 9A-9B middle. Whole blood sample control

[0100] Also disclosed herein is a synthetic whole blood sample control. In an embodiment, a synthetic whole blood sample control comprises (i) one or more cleavable hydrogel particle populations having an impedance substantially similar to that of a human red blood cell having an average diameter; (ii) a population of hemoglobin molecules or a population of dye molecules having an absorbance substantially similar to that of hemoglobin; and (iii) one or more non-cleavable hydrogel particle populations having an impedance substantially similar to that of a human white blood cell having an average diameter.

[0101] In an embodiment, the one or more non-cleavable hydrogel particle populations are synthetic leukocytes. In an embodiment, the non-cleavable hydrogel is composed of acrylamide and bisacrylamide. In an embodiment, the average diameter range of the one or more non-cleavable hydrogel particle populations is about 12 μm to about 22 μm to simulate monocytes. In an embodiment, the average diameter range of the one or more non-cleavable hydrogel particle populations is about 6 μm to about 18 μm to simulate lymphocytes. In an embodiment, the average diameter range of the one or more non-cleavable hydrogel particle populations is about 8 μm to about 15 μm to simulate neutrophils. In an embodiment, the average diameter range of the one or more non-cleavable hydrogel particle populations is about 12 μm to about 17 μm to simulate eosinophils. In an embodiment, the average diameter range of the one or more non-cleavable hydrogel particle populations is about 10 μm to about 15 μm to simulate basophils.

[0102] In embodiments, the synthetic leukocytes are synthetic monocytes. In embodiments, the synthetic leukocytes are synthetic granulocytes. In embodiments, the synthetic leukocytes are synthetic lymphocytes. In embodiments, the granulocytes are neutrophils, eosinophils, or basophils. In embodiments, the lymphocytes are T cells or B cells. Hydrogel

[0103] As described above, in the embodiments of the present disclosure, the engineered particles include multiple polymers or copolymers. In embodiments, the engineered particles include multiple hydrogel particles. Hydrogels are materials comprising a macromolecular three-dimensional network that allows them to swell when there is water, shrink in the absence of water (or by reducing the amount of water), but are insoluble in water. Swelling (i.e., absorption of water) is the result of the presence of hydrophilic functional groups attached to or dispersed in the macromolecular network. Crosslinking between adjacent macromolecules causes the water insolubility of these hydrogels. Crosslinking can be due to chemical bonds (i.e., covalent bonds) or physical bonds (i.e., van der Waals forces, hydrogen bonding, ionic forces, etc.). These chemical crosslinks can also be hydrolyzed under certain conditions, thereby reversing the insolubility of the hydrogel. Synthetic hydrogels can be prepared by polymerizing monomeric materials to form a skeleton and crosslinking the skeleton with a crosslinking agent. As mentioned herein, the term "hydrogel" refers to a macromolecular material in a dehydrated or hydrated state. The hydrogel with special value is characterized in that the material retains a general shape, whether it is dehydrated or hydrated. Thus, if a hydrogel has a roughly spherical shape under dehydrated conditions, it will be spherical under hydrated conditions.

[0104] In embodiments, the hydrogel particles disclosed herein comprise greater than about 30%, greater than about 40%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% water. In embodiments, the hydrogel particles have a water content of about 10% to about 95% by weight, or about 20% to about 95% by weight, or about 30% to about 95% by weight, or about 40% to about 95% by weight, or about 50% to about 95% by weight, or about 60% to about 95% by weight, or about 70% to about 95% by weight, or about 80% to about 95% by weight.

[0105] The hydrogel of particle form provided herein is synthesized by polymerizing one or more monomers provided herein.Synthesis is performed to form a single hydrogel particle.In an embodiment, a monomer material (monomer) is polymerized to form a homopolymer.However, in an embodiment, a copolymer (i.e., a comonomer) of different monomer units is synthesized, and it is used in the method provided herein.In an embodiment, the monomer or comonomer used in the methods and compositions described herein is a difunctional monomer or includes a difunctional monomer (in the case of using a comonomer).In an embodiment, a hydrogel is synthesized in the presence of a cross-linking agent.In another embodiment, a hydrogel is synthesized in the presence of a polymerization initiator.

[0106] The amount of monomer can be varied, for example, to obtain specific optical or morphological properties that are substantially similar to those of the target cells. In an embodiment, one or more monomer components (i.e., monomers, comonomers, difunctional monomers or combinations thereof, for example, N,N'-bis(acryloyl)cystamine, bis(2-methacryloyl)oxyethyl disulfide, allyl disulfide, polyethylene glycol (PEG) N-hydroxysuccinimide (NHS) ester disulfide, acryloyl-PEG-disulfide-PEG-acryloyl, 3-(2-pyridyldithio)propionic acid succinimidyl ester, diisopropylbenzene alcohol dimethacrylate, diisopropylbenzene alcohol diacrylate, 2,5-dimethyl-2,5-hexanediol dimethacrylate, acylhydrazone or 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane, allylamine or other comonomers or alginate providing chemical functionality for secondary labeling / conjugation at various cross-linking ratios) are present at about 10% by weight to about 95% by weight of the hydrogel. In further embodiments, the one or more monomer components are present at about 15% by weight to about 90% by weight of the hydrogel, or at about 20% by weight to about 90% by weight of the hydrogel.

[0107] Examples of various monomers and crosslinking chemistries that can be used with the present disclosure are provided in the Thermo Scientific Crosslinking Technical Handbook entitled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf), the disclosure of which is incorporated by reference in its entirety for all purposes. For example, hydrazine (e.g., with NHS ester compounds) or EDC coupling reactions (e.g., with maleimide compounds) can be used to construct the hydrogels of the present disclosure.

[0108] In embodiments, monomers for use with the hydrogels provided herein are lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, derivatized forms thereof, or combinations thereof.

[0109] In an embodiment, one or more of the following monomers are used herein to form the hydrogels of the present disclosure: 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glyceryl methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, or a combination thereof.

[0110] In embodiments, one or more of the following monomers are used herein to form tunable hydrogels: phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachloro ... Acid ester, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenyl acrylamide, N-phenyl Methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-dibenzyl methacrylamide, N-(4-methylphenyl) methacrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl) acrylamide, N-trityl acrylamide, N-(4-hydroxyphenyl) acrylamide, N,N-methylphenyl acrylamide, N,N-phenylphenethyl acrylamide, N-dibenzyl methacrylamide, N-(

[00136] The invention also includes but is not limited to the following: methacrylamide, N-(4-methylphenyl)methyl methacrylamide, N-1-naphthyl methacrylamide, N-4-nitrophenyl methacrylamide, N-(2-phenylethyl) methacrylamide, N-trityl methacrylamide, N-(4-hydroxyphenyl) methacrylamide, N,N-methylphenyl methacrylamide, N,N'-phenylphenethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, as described in U.S. Pat. No. 6,657,030, which is incorporated herein by reference in its entirety for all purposes.

[0111] Both synthetic monomers and biological monomers can be used in hydrogels provided herein to form synthetic hydrogels, biological hydrogels or hybrid hydrogels comprising synthetic components and biological components (e.g., peptides, proteins, monosaccharides, disaccharides, polysaccharides, primary amines, sulfhydryl groups, carbonyl groups, carbohydrates, carboxylic acids present on biomolecules). For example, proteins, peptides or carbohydrates can be used as separate monomers to form hydrogels, and the hydrogels include or do not include synthetic monomers (or polymers) and chemically compatible comonomers and crosslinking chemicals (see, for example, Thermo Scientific Crosslinking Technology Handbook entitled "Easy Molecular Bonding Crosslinking Technology", available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf, and its disclosure is incorporated by reference in its entirety for all purposes). Compatible crosslinking chemicals include, but are not limited to, amines, carboxyls and other reactive chemical side groups. Representative reactive groups suitable for use in hydrogels and monomers described herein are provided in Table 2 below. Table 2. Cross-linker reactive groups suitable for biomonomer conjugation

[0112] Generally, any form of polymerization chemistry / method known to those skilled in the art can be used to form the polymer. In embodiments, polymerization can be catalyzed by ultraviolet light induced free radical formation and reaction. In other embodiments, the hydrogel particles of the present disclosure are produced by polymerization of acrylamide or polymerization of acrylates. For example, in embodiments, the acrylamide is a polymerizable carbohydrate-derivatized acrylamide as described in U.S. Pat. No. 6,107,365, the disclosure of which is incorporated by reference in its entirety for all purposes. As described therein and known to those of ordinary skill in the art, the specific attachment of acrylamide groups to sugars is readily adapted to a range of monosaccharides and higher order polysaccharides, such as synthetic polysaccharides or polysaccharides derived from natural sources, such as glycoproteins found in serum or tissues.

[0113] In embodiments, acrylate-functionalized poly(ethylene glycol) monomers are used as hydrogel monomers. For example, in embodiments, the PEG is an acrylate- or acrylamide-functionalized PEG.

[0114] In embodiments, the hydrogel particles include monofunctional monomers polymerized with at least one difunctional monomer. An example includes, but is not limited to, the use of acrylamide and bisacrylamide (difunctional monomers) to form polyacrylamide polymers. In embodiments, the hydrogel particles provided herein include difunctional monomers polymerized with a second difunctional monomer. An example includes, but is not limited to, the formation of polymers having a mixed composition containing compatible chemicals, such as acrylamide, bisacrylamide, and bisacrylamide structural homologs containing a wide range of other chemicals. The scope of chemically compatible monomers, difunctional monomers, and mixed compositions is clear to those skilled in the art and follows the principles of chemical reactivity known to those skilled in the art. (References Thermo Manual and Acrylamide Polymerization Handbook). See, e.g., the Thermo Scientific Crosslinking Technical Handbook entitled “Easy molecular bonding crosslinking technology” (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf) and the Polyacrylamide Emulsions Handbook (SNF Floerger, available at snf.com.au / downloads / Emulsion_Handbook_E.pdf), the disclosures of each of which are incorporated by reference in their entirety for all purposes.

[0115] In an embodiment, the hydrogel particles provided herein include a polymerizable monofunctional monomer (and is a monofunctional acrylic monomer). Non-limiting examples of monofunctional acrylic monomers for use herein are acrylamide; methacrylamide; N-alkyl acrylamides, such as N-ethyl acrylamide, N-isopropyl acrylamide or N-tert-butyl acrylamide; N-alkyl methacrylamides, such as N-ethyl methacrylamide or N-isopropyl methacrylamide; N,N-dialkyl acrylamides, such as N,N-dimethyl acrylamide and N,N-diethyl-acrylamide; N-[(dialkylamino)alkyl]acrylamide, such as N-[3-dimethylamino)propyl]acrylamide; amine or N-[3-(diethylamino)propyl]acrylamide; N-[(dialkylamino)alkyl]methacrylamide, such as N-[3-dimethylamino)propyl]methacrylamide or N-[3-(diethylamino)propyl]methacrylamide; (dialkylamino)alkyl acrylates, such as 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)propyl acrylate or 2-(diethylamino)ethyl acrylate; and (dialkylamino)alkyl methacrylates, such as 2-(dimethylamino)ethyl methacrylate.

[0116] A bifunctional monomer is any monomer that can be polymerized with a monofunctional monomer of the present disclosure to form a hydrogel as described herein, which further contains a second functional group that can participate in a second reaction (e.g., conjugation of a fluorophore or a cell surface receptor (or a domain thereof)).

[0117] In an embodiment, the difunctional monomer is selected from the group consisting of: allylamine, allyl alcohol, allyl isothiocyanate, allyl chloride, and allyl maleimide.

[0118] The difunctional monomer may be a difunctional acrylic monomer. Non-limiting examples of difunctional acrylic monomers are N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, N,N'-ethylenebisacrylamide, N,N'-ethylenebismethacrylamide, N,N'-bisacryloylcystamine, N,N'-propylenebisacrylamide and N,N'-(1,2-dihydroxyethylene)bisacrylamide.

[0119] In an embodiment, the hydrogel comprises the residue of a bifunctional monomer, wherein the bifunctional monomer allows the hydrogel to be cleaved in the presence of a lysis buffer. In an embodiment, the bifunctional monomer comprises a bond that can be cleaved by a lysis buffer. In an embodiment, the bifunctional monomer comprises a disulfide bond. In an embodiment, the bifunctional monomer comprising a disulfide bond is N, N'-bis (acryloyl) cystamine, bis (2-methacryloyl) oxyethyl disulfide, allyl disulfide, polyethylene glycol (PEG) N-hydroxysuccinimide (NHS) ester disulfide, acryloyl-PEG-disulfide-PEG-acryloyl or 3-(2-pyridyl dithio) propionic acid succinimide ester. In an embodiment, the bifunctional monomer comprises an acid labile bond. In an embodiment, the difunctional monomer is diisopropylbenzene alcohol dimethacrylate, diisopropylbenzene alcohol diacrylate, 2,5-dimethyl-2,5-hexanediol dimethacrylate, acylhydrazone, or 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane. In an embodiment, the acylhydrazone has In an embodiment, the bifunctional monomer comprises an enzyme-degradable bond. In an embodiment, the bifunctional monomer is

[0120] Higher order branched and linear comonomers in the polymer blend can be substituted to tailor the refractive index while maintaining polymer density as described in US Pat. No. 6,657,030 (incorporated herein by reference in its entirety for all purposes).

[0121] In embodiments, the biomonomer is functionalized with acrylamide or acrylate. For example, in embodiments, the polymerizable acrylamide-functionalized biomolecule is an acrylamide- or acrylate-functionalized protein (e.g., an acrylamide-functionalized collagen or a functionalized collagen domain), an acrylamide- or acrylate-functionalized peptide, or an acrylamide- or acrylate-functionalized monosaccharide, disaccharide, or polysaccharide.

[0122] Any monosaccharide, disaccharide or polysaccharide (functionalized or otherwise) can be used as a hydrogel monomer. In an embodiment, a monosaccharide, disaccharide or polysaccharide functionalized with acrylamide or acrylate is used as a polymerizable hydrogel monomer. In an embodiment, a structural polysaccharide is used as a polymerizable hydrogel monomer. In another embodiment, the structural polysaccharide is arabinoxylan, cellulose, chitin or pectin. In an embodiment, alginic acid (alginate) is used as a polymerizable hydrogel monomer. In another embodiment, glycosaminoglycan (GAG) is used as a polymerizable monomer in the hydrogel provided herein. In another embodiment, GAG is chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate or hyaluronic acid (hyaluronic acid) (also referred to as hyaluronic acid (hyaluron) or hyaluronic acid (hyaluronate) in the art), which is used as a polymerizable hydrogel monomer. Other compatible biomonomers and reactive chemicals thereof are known to those skilled in the art, and follow general chemical reactivity principles.

[0123] In embodiments, biocompatible monomers for use with the hydrogels described herein include ethylene glycol dimethacrylate (EGDMA), 2-hydroxyethyl methacrylate (HEMA), methyl methacrylate (MMA), methacryloxymethyltrimethylsilane (TMS-MA), N-vinyl-2-pyrrolidone (N-VP), styrene, or a combination thereof.

[0124] Naturally occurring hydrogels that can be used in the present disclosure include various polysaccharides that can be obtained from natural sources (such as plants, algae, fungi, yeasts, marine invertebrates and arthropods). Non-limiting examples include agarose, dextran, chitin, cellulose-based compounds, starch, derivatized starch, etc. These will generally have repeated glucose units as the main part of the polysaccharide backbone. The cross-linking chemicals of such polysaccharides are known in the art, see, for example, Thermo Scientific Crosslinking Technical Handbook entitled "Easymolecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf).

[0125] In an embodiment, hyaluronic acid is used as a hydrogel monomer (as a single monomer or as a comonomer). In an embodiment, hyaluronic acid is functionalized, for example, by acrylate or acrylamide. Hyaluronic acid is a high molecular weight GAG consisting of disaccharide repeating units of N-acetylglucosamine and glucuronic acid linked together by alternating β-1,4 and β-1,3 glycosidic bonds. In the human body, hyaluronic acid is present in several soft connective tissues, including skin, umbilical cord, synovial fluid and vitreous fluid. Therefore, in an embodiment where it is necessary to simulate one or more optical properties of skin cells, umbilical cord cells or vitreous fluid cells, in an embodiment, hyaluronic acid is used as a hydrogel monomer. The method for making hydrogel particles is described in Xu et al. (2012). Soft Matter.8, pp. 3280-3294, and its disclosure is incorporated herein in its entirety for all purposes. As described therein, hyaluronic acid can be derivatized with various reaction pathways, depending on the desired cross-linking chemicals and other monomers for forming hydrogel particles.

[0126] In yet other embodiments, chitosan, a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units), is used as a hydrogel monomer (either as a single monomer or as a comonomer).

[0127] Other polysaccharides for use as monomers or co-monomers in hydrogels include, but are not limited to, agar, agarose, alginic acid, polysaccharide acid, alpha glucan, pullulan, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan polysaccharides (e.g., of the kappa, iota or lambda variety), cellodextrin, animal cellulose, cellulose, chitin, chitosan, chrysophyllin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, polysaccharide, levan, fucoidan, galactoglucomannan, galactomannans, galactosaminogalactans , gellan gum, glucan, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentamer, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scallion, sizoran, welan gum, xanthan gum, xylan, xyloglucan, zymosan or a combination thereof. As described throughout, the polysaccharide may be further functionalized depending on the desired cross-linking chemistry and / or additional comonomers employed in the hydrogel. For example, in an embodiment, one or more polysaccharides described herein are functionalized with acrylate or acrylamide.

[0128] In embodiments, a single hydrogel particle or a plurality thereof comprises a peptide, a protein, a protein domain or a combination thereof as a hydrogel monomer or a plurality thereof. In further embodiments, the protein is a structural protein or a domain thereof, such as, for example, silk protein, elastin, titin or collagen or a domain thereof. In embodiments, the protein is an extracellular matrix (ECM) component (e.g., collagen, elastin, proteoglycan). In even further embodiments, the structural protein is collagen. In yet further embodiments, the collagen is type I collagen, type II collagen or type III collagen or a combination thereof. In embodiments, the hydrogel monomer comprises a proteoglycan. In further embodiments, the proteoglycan is decorin, biglycan, testisan, bikunitz inhibitor, fibromodulin, lumican or a domain thereof.

[0129] In an embodiment, acrylate-functionalized structural protein hydrogel monomers (e.g., acrylate-functionalized proteins or protein domains, such as silk protein, elastin, titin, collagen, proteoglycans, or functionalized domains thereof) are used as components of the hydrogels provided herein. In additional embodiments, acrylate-functionalized structural protein hydrogel monomers include proteoglycans, such as decorin, biglycan, testoglycan, bikunitz inhibitor, fibromodulin, lumican, or domains thereof.

[0130] In an embodiment, PEG monomers and oligopeptides that can simulate extracellular matrix proteins are used in the hydrogels provided herein, such as multi-arm PEG, integrin binding peptides and double cysteine ​​matrix metalloproteinase peptides functionalized with vinyl sulfone, such as Lutolf et al. (2003). Proc. Natl. Acad. Sci. USA 100, 5413-5418 (incorporated by reference in its entirety for all purposes). In this specific embodiment, a hydrogel is formed by a Michael-type addition reaction between dithiolated oligopeptides and the vinyl sulfone groups on PEG. The scope of other compatible chemicals that can be incorporated here is clear to those skilled in the art, and follows general chemical reactivity principles, see, for example, Thermo Scientific Crosslinking Technical Handbook entitled "Easymolecular bonding crosslinking technology" (available at tools. lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf).

[0131] Other biologically active domains in natural proteins can also be used as hydrogel monomers or parts thereof. For example, cell adhesion integrin binding domains, controlled release affinity binding domains, or transglutaminase cross-linking domains can be used in the hydrogels provided herein. Details for producing such hydrogels can be found in Martino et al. (2009). Biomaterials 30, 1089; Martino et al. (2011). Sci. Trans. Med. 3, 100ra89; Hu and Messersmith (2003). J. Am. Chem. Soc. 125, 14298, each of which is incorporated by reference in its entirety for all purposes.

[0132] In embodiments, recombinant DNA methods are used to produce proteins that are designed to gel in response to changes in pH or temperature, such as described by Petka et al. (1998). Science 281, pp. 389-392 (incorporated by reference in its entirety for all purposes). Briefly, the protein consists of terminal leucine zipper domains flanking water-soluble polyelectrolyte segments. In near-neutral aqueous solutions, coiled-coil aggregates of the terminal domains form a three-dimensional hydrogel polymer network.

[0133] A further range of biocompatible monomers that may be incorporated are known in the art, see for example the non-degradable biocompatible monomers disclosed in Shastri (2003). Current Pharmaceutical Biotechnology 4, pp. 331-337, which is incorporated herein by reference in its entirety for all purposes. Further monomers are provided in de Moraes Porto (2012). Polymer Biocompatibility, Polymerization, Dr. Ailton De Souza Gomes (ed.), ISBN: 978-953-51-0745-3; InTech, DOI: 10.5772 / 47786; Heller et al. (2010). Journal of Polymer Science Part A: Polymer Chemistry 49, pp. 650-661; Final Report for Biocompatible Materials (2004), The Board of the Biocompatible Materials and the Molecular Engineering in Polymer Science programmes, ISBN 91-631-4985-0, the disclosures of each of which are hereby incorporated by reference in their entirety. Cross-linking

[0134] Common crosslinking agents that can be used to crosslink the hydrogels provided herein include, but are not limited to, ethylene glycol dimethacrylate (EGDMA), tetraethylene glycol dimethacrylate, bis(2-methacryloyl)oxyethyl disulfide, and N,N'-15 methylenebisacrylamide. The range of additional crosslinking chemistries that can be used is clear to those skilled in the art and follows general chemical reactivity principles, see, for example, Thermo Scientific Crosslinking Technical Handbook entitled "Easy molecular bondingcrosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf).

[0135] In an embodiment, the polymerization of the hydrogel is initiated by an equivalent initiator formed by a persulfate or catalytic free radical. The scope of compatible initiators is known to those skilled in the art and follows general chemical reactivity principles, see, for example, Thermo Scientific Crosslinking Technical Handbook entitled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf). Persulfate can be any water-soluble persulfate. Non-limiting examples of water-soluble persulfates are ammonium persulfate and alkali metal persulfates. Alkali metals include lithium, sodium and potassium. In an embodiment, persulfate is ammonium persulfate or potassium persulfate. In another embodiment, the polymerization of the hydrogel provided herein is initiated by ammonium persulfate.

[0136] The polymerization of the hydrogel can be promoted by a promoter, which can catalyze the formation of unstable chemical side groups. The range of possible promoters is known to those skilled in the art, and follows general chemical reactivity principles, see, for example, ThermoScientific Crosslinking Technical Handbook entitled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf). In an embodiment, the promoter is a tertiary amine. The tertiary amine can be any water-soluble tertiary amine. In an embodiment, the promoter is used in the polymerization reaction and is N, N, N', N'-tetramethylethylenediamine, 3-(dimethylamino) propionitrile or N, N, N', N'-tetramethylethylenediamine (TEMED). In an embodiment, the promoter is used in the polymerization reaction and is azobis(isobutyronitrile) (AIBN).

[0137] As discussed above, hydrogels for use in the compositions and methods described herein can comprise any monomer units and crosslinkers as described herein, and in one aspect, are produced as hydrogel particles by polymerizing droplets (see, e.g., Figure 2). Microfluidic methods of generating a plurality of droplets (including fluid droplets and hardened droplets) are known to those of ordinary skill in the art and are described in U.S. Patent Publication No. 2011 / 0218123 and U.S. Patent No. 7,294,503, each of which is incorporated herein by reference in its entirety for all purposes. Such methods provide a plurality of droplets containing a first fluid and substantially surrounded by a second fluid, wherein the first fluid and the second fluid are substantially immiscible (e.g., droplets containing a water-based liquid are substantially surrounded by an oil-based liquid). Cleavable

[0138] A complete blood count (CBC) is a blood test used to evaluate health and detect various disorders, including anemia, infection, and leukemia. The CBC test measures several components, including: red blood cells, white blood cells, hemoglobin, hematocrit, and platelets. Red blood cells in a sample are typically lysed to measure the hemoglobin in the sample. Therefore, there is a need for a control that can be lysed itself.

[0139] Lysis buffer can use ammonium chloride. Blood lysis buffer usually uses ammonium chloride, including 1X RBC lysis buffer and 10X RBC lysis buffer from ThermoFisher Scientific. The blood lysis buffer for clinical blood samples is designed to lyse anucleated red blood cells and preserve white blood cells to perform quantitative measurement of white blood cell count and hemoglobin. Other lysis buffers can be designed to dissolve engineered particles, and these other lysis buffers include strong reducing agents (such as dithiothreitol (DTT) tris (2-carboxyethyl) phosphine (TCEP), beta-mercaptoethanol (BME) or glutathione (GSH)). Other lysis buffers can be designed to dissolve acid unstable bonds. For example, the lysis buffer can include formic acid. Other lysis buffers can be designed to dissolve enzyme degradable bonds. For example, the lysis buffer can include chymotrypsin. Other non-limiting examples include divalent ions, such as ethylenediaminetetraacetic acid (EDTA) or citrate.

[0140] In embodiments, individual particles or a plurality thereof include biodegradable polymers as monomers. In embodiments, biodegradable polymers are poly(esters) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), polylactic co-glycolic acid (PLGA) and copolymers thereof. These polymers can be degraded by hydrolysis, thereby dissolving macromolecular particles. In embodiments, biodegradable polymers are carbohydrates or proteins or combinations thereof. For example, in embodiments, monosaccharides, disaccharides or polysaccharides (e.g., glucose, sucrose or maltodextrin), peptides, proteins (or their domains) are used as hydrogel monomers. Other biodegradable polymers include poly(hydroxyalkanoates) of PHB-PHV categories, additional poly(esters) and natural polymers, such as modified poly(sugars), such as starch, cellulose and chitosan. In embodiments, biocompatible polymers are adhesive proteins, cellulose, carbohydrates, starch (e.g., maltodextrin, 2-hydroxyethyl starch, alginic acid), dextran, lignin, polyamino acids, amino acids or chitin. Such biodegradable polymers are commercially available, for example, from Sigma Aldrich (St. Louis, MO).

[0141] In an embodiment, the protein comprises only natural amino acids. However, the present disclosure is not limited thereto. For example, self-assembling artificial proteins and proteins with non-natural amino acids (e.g., those incorporated into non-ribosomal peptides or introduced via synthetic methods, see, for example, Zhang et al. (2013). Current Opinion in Structural Biology 23, pp. 581-587, the disclosure of which is incorporated by reference in its entirety for all purposes) or its protein domains can also be used as hydrogel monomers. The scope of non-natural (non-natural) (non-natural (unnatural)) amino acids that can be incorporated into such compositions is well known to those skilled in the art (Zhang et al. (2013). Current Opinion in Structural Biology 23, pp. 581-587; it is incorporated by reference in its entirety for all purposes). In an embodiment, a biodegradable polymer is used as a comonomer, i.e., a mixture of monomers. In an embodiment, the biodegradable polymer is a difunctional monomer.

[0142] In an embodiment, a single hydrogel particle or a plurality thereof comprises a degradable polymer as a hydrogel monomer. In an embodiment, the degradable polymer is a poly(ester) based on PLA, PGA, PCL, PLGA and copolymers thereof. In an embodiment, the degradable polymer is based on any of the monomers described herein, which can be degraded by mechanical degradation, chemical degradation, combinations thereof, or by any other degradation mechanism. For example, the monomer can be acrylamide, and the degradable polymer formed from acrylamide can be degraded by exposure to potassium persulfate.

[0143] In embodiments, degradation of an individual hydrogel particle or a plurality thereof (whether by biodegradable means, lysis, or otherwise) can result in the release of a substance contained therein. For example, when the substance is a biomolecule such as hemoglobin, degradation of hydrogel particles comprising hemoglobin can allow for measurement of hemoglobin separated from hydrogel monomers. In another instance, when the substance is a biomolecule such as hemoglobin, degradation of hydrogel particles comprising hemoglobin can be performed by a lysis buffer.

[0144] A plurality of fluid droplets (e.g., prepared using a microfluidic device) can be polydisperse (e.g., having a range of different sizes), or in some cases, the fluid droplets can be monodisperse or substantially monodisperse, e.g., having a uniform distribution of diameters, e.g., such that no more than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the droplets have an average diameter that is greater than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the average diameter. As used herein, the average diameter of a population of droplets refers to the arithmetic mean of the droplet diameters. The average diameter of the particles can be measured, for example, by light scattering techniques. In embodiments, the average diameter of the hydrogel particles is customized, for example, by changing the flow rate of the first and second fluids within one or more channels of the microfluidic device, or by changing the volume of one or more channels of the microfluidic device.

[0145] Thus, the present disclosure provides a population of hydrogel particles comprising a plurality of hydrogel particles, wherein the population of hydrogel particles is substantially monodisperse.

[0146] The term microfluidics refers to a device, apparatus or system comprising at least one fluid channel having a cross-sectional dimension of less than 1 mm and a ratio of length to the largest cross-sectional dimension perpendicular to the channel of at least about 3: 1. Microfluidic devices comprising microfluidic channels are particularly suitable for preparing a plurality of monodisperse droplets.

[0147] Non-limiting examples of microfluidic systems that can be used with the present disclosure are disclosed in U.S. Patent Application Publication No. 2006 / 0163385; U.S. Patent Application Publication No. 2005 / 0172476; U.S. Patent Application Publication No. 2007 / 000342; International Patent Application Publication No. WO 2006 / 096571; U.S. Patent Application Publication No. 2007 / 0054119; U.S. Patent No. 7,776,927; and International Patent Application Publication No. WO 2006 / 078841, each of which is incorporated herein by reference in its entirety for all purposes. Functionalization (encapsulated, on the surface and / or removable)

[0148] In embodiments, a bead, a plurality of beads, a biomolecule, or a plurality of biomolecules are embedded (encapsulated) within a hydrogel particle. In embodiments, the encapsulated bead or biomolecule is used to simulate one or more intracellular organelles of a target cell, or to simulate a cell after engulfing a particle. In embodiments, encapsulation or embedding of the bead or biomolecule is completed when the hydrogel particle is formed. For example, the bead can be suspended at an appropriate concentration to allow an average of one bead to be embedded / encapsulated in a single hydrogel particle. The bead suspension can be contained in an aqueous solution of, for example, a monomer. Similarly, a biomolecule or a mixture of biomolecules can be incorporated into an aqueous solution of a monomer to encapsulate one or more biomolecules.

[0149] Alternatively, once the hydrogel particles are formed, for example by the methods described above, in embodiments, they may be further manipulated, for example by embedding a bead, a plurality of beads, a biomolecule, or a plurality of biomolecules within the hydrogel particles.

[0150] Thus, in one aspect of the present disclosure, a hydrogel comprising an embedded substance is provided.

[0151] In an embodiment, the embedding material is an embedding molecule, such as a biomolecule. The biomolecule can be a single species or a variety of different species. For example, a protein, a peptide, a carbohydrate, a nucleic acid or a combination thereof can be encapsulated in the hydrogel particles of the present disclosure. In addition, different nucleic acid molecules (for example, with different sequences or nucleic acid types, such as genomic DNA, messenger RNA or DNA-RNA hybrids) can be encapsulated by the hydrogel particles of the present disclosure. These can be composed of any protein or nucleic acid, because both forms of biomaterials contain unstable chemical side groups or can be modified by commercial suppliers (for example, Integrated DNA Technology chemical side group modification). Such side groups are compatible with common reaction chemicals in comonomer compositions (for example, acrylate chemicals, NHS esters, primary amines, copper-catalyzed click chemistry (Sharpless)). The scope of possible embedding molecules containing compatible chemicals is understood by those skilled in the art. In an embodiment, the embedded molecule can be one or more polynucleotides or analogs thereof. In an embodiment, the embedded molecule can be one or more polypeptides or analogs thereof. In an embodiment, the embedded molecule can be a complex of one or more polynucleotides and one or more polypeptides or analogs thereof. In embodiments, the entrapped molecule may be one or more polynucleotides, one or more polypeptides, a complex of one or more polynucleotides and one or more polypeptides, or analogs thereof.

[0152] In embodiments, when the target cell is a red blood cell, the biomolecule embedded in the particle can be hemoglobin or a hemoglobin-like molecule, a dye or an oxygen-carrying molecule, etc. Hemoglobin can be embedded in the hydrogel and combined with the hydrogel. In the case where the hydrogel is an amine-functionalized acrylamide, hemoglobin can react with the modified amine groups of the hydrogel via the sulfhydryl (i.e., thiol group) of the cysteine ​​of hemoglobin. The hydrogel crosslinked with the thiol group of hemoglobin can be prepared by treating with a heterobifunctional crosslinking agent containing both succinimide ester and maleimide. Maleimide-modified biomolecules can then be reacted with thiol-containing hemoglobin to form a stable thioether crosslink.

[0153] In embodiments, any one or more of hemoglobin or hemoglobin-like molecules, dyes or oxygen carrier molecules, oxygen transport molecules, etc. can be on the surface of the engineered particles. In embodiments, the engineered particles contain one or more functional groups that can be used for the attachment of any one or more of hemoglobin or hemoglobin-like molecules, dyes or oxygen carrier molecules, oxygen transport molecules, etc. In embodiments, the functional groups are amine groups, carboxyl groups, hydroxyl groups, or combinations thereof. Depending on the desired functionalization, it will be appreciated that a variety of bifunctional monomers can be used, for example, using different chemistries and different molecules to functionalize the particles.

[0154] In an embodiment, any one or more of hemoglobin or hemoglobin-like molecules, dyes or oxygen carrying molecules, oxygen transport molecules, etc. can be in solution with the engineered particles. In this embodiment, the engineered cell population simulates and can be a control for a CBC test, where the red blood cells have been lysed and the hemoglobin is no longer within the red blood cells. Subgroup

[0155] In an embodiment, different engineered particle subpopulations are manufactured, each with a different concentration of biomolecules. In another embodiment, the biomolecule is any one or more of hemoglobin or hemoglobin-like molecules, dyes or oxygen carrier molecules, oxygen transport molecules, etc. In another embodiment, the biomolecule is a nucleic acid, a protein, an intracellular ion, such as calcium acid (or other biomolecules selected by the user, such as calcium). In an embodiment, different hydrogel particle subpopulations are manufactured, each with a different concentration of drug substances. In an embodiment, the drug substance is a biomolecule (i.e., a biologic, an antibody, an antibody drug conjugate, a protein / enzyme, a peptide, a non-ribosomal peptide, or a related molecule) or a small molecule synthetic drug (e.g., type I / II / III polyketides, non-ribosomal peptides with biologically active properties, or other small molecule entities generally classified by those skilled in the art).

[0156] In this respect, the present disclosure is particularly useful for determining the assay resolution, wherein cells are dyed to measure their corresponding nucleic acid or protein content. In embodiments, different engineered particle populations provided herein are encapsulated with any one or more of known, different amounts of hemoglobin or hemoglobin-like molecules, dyes or oxygen carrier molecules, oxygen transport molecules, etc. For intracellular material, separate engineered particles are dyed, and the fluorescence of the separate particles of each colony is measured via a hematology analyzer. This allows the generation of a standard curve to determine the sensitivity and dynamic range of the intracellular assay. After establishment, samples can be passed through a cell counter to detect one or more target cells (if present), and the amount of the intracellular material in the corresponding one or more target cells is quantitatively measured. In embodiments, the embedded material is hemoglobin.

[0157] In an embodiment, the methods provided herein are used to determine the sensitivity and / or dynamic range of a hematology quantitative assay. In this embodiment, the sample is interrogated for the cell type (if present) within the sample and the amount of intracellular biomolecules.

[0158] In an embodiment, the present disclosure provides a means for determining the resolution and / or sensitivity of a hematology assay. In an embodiment, hydrogel particles encapsulate known amounts of any one or more of hemoglobin or hemoglobin-like molecules, dyes or oxygen-carrying molecules, oxygen-transporting molecules, etc., at various concentrations, and can be subsequently stained with an appropriate stain. The fluorescence of each particle is measured to determine the sensitivity and / or dynamic range of the assay. The fluorescence value can then be compared to the value obtained from the cells in the sample to determine whether a target cell is present and whether it contains intracellular protein, as well as the amount of protein.

[0159] In embodiments, individual hydrogel particles are tuned to have at least one optical property and / or morphological property similar to that of red blood cells. Individual particles are embedded with known amounts of a biomolecule of interest (e.g., hemoglobin). The particles are used to generate a standard curve for a biomolecule detection assay for a specific cell type. method

[0160] In an embodiment, a method for calibrating a hematology analyzer for analyzing target cells is provided. In an embodiment, the method comprises: (a) inserting a red blood cell control composition having properties substantially similar to the properties of the target cells into a device; b) measuring the properties of the red blood cell control composition using the hematology analyzer; c) comparing the measured properties of the red blood cell control composition with the target sample; and b) optionally adjusting the hematology analyzer so that the properties of the red blood cell control composition are substantially similar to the properties of the target sample, thereby calibrating the hematology analyzer for analyzing human whole blood samples.

[0161] In an embodiment, a method for evaluating a red blood cell sample using a hematology analyzer is provided. In an embodiment, the method comprises (a) measuring one or more properties of a red blood cell control composition, (b) measuring one or more properties of the red blood cell sample; and (c) comparing the measurement results of the red blood cell sample with the measurement results of the control sample.

[0162] In an embodiment, the one or more cleavable hydrogel particle populations have characteristics of normal red blood cells, as disclosed in the "Red Blood Cell Control" section of this application. In an embodiment, a deviation in the measurement results of the red blood cell sample from the measurement results of the red blood cell control composition indicates a condition or disease.

[0163] In an embodiment, the one or more cleavable hydrogel particle populations have characteristics of red blood cells in a disease state, as disclosed in the "Red Blood Cell Control" section of this application. In an embodiment, a substantial similarity between the measurement results of the red blood cell sample and the measurement results of the control sample indicates a condition or disease.

[0164] Hematology analyzers are known in the art and include commercially available devices for performing flow cytometry. Hematology analyzers can employ, for example, hole-based techniques, image-based techniques, and / or waveform-based techniques, etc. In embodiments, hematology analyzers are optical-based cell counters. In embodiments, hematology analyzers are resistance-based cell counters. Example

[0165] The present disclosure is further illustrated by reference to the following examples. However, it should be noted that these examples, like the embodiments described above, are illustrative and are not to be construed in any way as limiting the scope of the present disclosure. Example 1: Production of hydrogel particles

[0166] Photomasks for UV lithography were from CADart Services Inc. and designed using AutoCad (AutoDesk, inc.). SU-8 photoresist (Microchem, inc.) was photocrosslinked on a 4" silicon wafer using a collimated UV light source (OAI, inc.) to create a master for microfluidic device fabrication. PDMS (polydimethylsiloxane, Sigma Aldrich, inc.) was prepared and formed using standard published methods for soft lithography and microfluidic device fabrication (see McDonald JC et al., 2000, Electrophoresis 21: 27-40).

[0167] Droplets were formed using flow focusing geometry, where two oil channels focused the central flow of the aqueous monomer solution to break up the droplets in the water-in-oil emulsion. Fluorinated hydrocarbon oil (Novec 7500 3M, inc.) was used as the external continuous phase liquid for droplet formation. To stabilize the droplets prior to polymerization, 0.5% w / w of surfactant was added to the oil phase (Krytox 157 FSH, ammonium carboxylate salt from Dupont). To prepare the basic polyacrylamide gel particles, a central phase containing an aqueous monomer solution of the following is used: N-acrylamide (1%-20% w / v), a cross-linker that allows hydrogel cleavage (N,N'-bis(acryloyl)cystamine, bis(2-methacryloyl)oxyethyl disulfide, allyl disulfide, polyethylene glycol (PEG) N-hydroxysuccinimide (NHS) ester disulfide, acryloyl-PEG-disulfide-PEG-acryloyl, or 3-(2-pyridyldithio)propionic acid succinimidyl ester, diisopropylbenzene alcohol dimethacrylate, diisopropylbenzene alcohol diacrylate, 2,5-dimethyl-2,5-hexanediol dimethacrylate, acylhydrazone or 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane), an accelerator and ammonium persulfate (1% w / v). An accelerator (N,N,N',N'-tetramethylethylenediamine, 2% vol%) was added to the oil phase to initiate polymerization of the hydrogel particles after droplet formation.

[0168] Comonomers can be added to the base gel formulation to increase functionality. Allyl-amines provide primary amine groups for secondary labeling after gel formation. Forward scattering can be adjusted by adjusting the refractive index of the gel by adding comonomers allyl acrylate and allyl methacrylate. Side scattering of droplets can be adjusted by adding a colloidal suspension of silica nanoparticles and / or PMMA (poly(methyl methacrylate)) particles (about 100 nm) to the central aqueous phase before polymerization.

[0169] Stoichiometric multiplexing of hydrogel particles is achieved by secondary labeling using comonomers containing chemically orthogonal side groups (amine, carboxyl, maleimide, epoxide, alkyne, etc.).

[0170] Droplets were formed at an average rate of 5 kHz and collected in the fluorocarbon oil phase.Polymerization was completed at 50°C for 30 minutes and the resulting hydrogel particles were washed from the oil into aqueous solution. Example 2: Production and visualization of non-cleavable hydrogel particles

[0171] Water containing 5% acrylamide, 0.25% bisacrylamide, 0.05% allylamine, and 0.1% ammonium persulfate was flowed through the central channel and aggregated through oil containing 0.1% TEMED and passed through a 10 μm nozzle to produce 10 μm hydrogel particles, as shown in FIG. Figure 3A After polymerization, the particles were washed in water, as Figure 3B As shown, and conjugated with the desired dye. The fluorescent hydrogel particles were visualized by fluorescence microscopy, as shown Figure 3C The size of the hydrogel can be adjusted by increasing or decreasing the nozzle size. Example 3: Generation of RBC mimics

[0172] The hydrogel particles can be adjusted to different sizes and / or polymer contents. Figure 7B-7D As shown, exemplary RBC mimics have a Coulter volume corresponding to a diameter in a range between 3 μm and 8 μm; a Coulter volume corresponding to a diameter of about 12 μm, but with a low polymer weight percentage (this reduces distribution); or a Coulter volume corresponding to a diameter of about 12 μm, but with a high polymer weight percentage (this increases distribution). As measured by the Coulter principle, the Coulter volume can be considered as a perceived volume. However, the actual diameter of each of these RBC mimics can be different depending on the porosity of the engineered particles (indicated by the weight percentage of the polymer).

[0173] Different sizes and / or polymer contents of hydrogel particles can mimic different disease patterns. Figure 8A-8B Additional examples are shown in which the RBC mimic is adjusted to simulate microcytic RBCs, macrocytic RBCs, or anisocytic RBCs. Example 4: Production of HGB mimetics

[0174] A red dye (Allura Red or any dye with a spectrum similar to that of hemoglobin) can be used to produce a human HGB mimetic. Aqueous solutions of Allura Red at different concentrations were prepared and analyzed using a Beckman Coulter DxH690T hematology analyzer. Fig.9A As shown, there is a linear relationship between the concentration of Allura Red and the concentration of human HGB, allowing accurate reproduction of any specific hemoglobin-related disease. At 5 mg / mL of Allura Red, the results from the analyzer are similar to those of normal human whole blood. At low concentrations of Allura Red, the results are similar to those of patients with too little hemoglobin or anemia. At high concentrations of Allura Red, the results are similar to those of patients with too much hemoglobin.

[0175] HGB from animals other than humans can also be used to generate human HGB mimetics. Bovine HGB solutions of varying concentrations were prepared and analyzed using a Beckman Coulter DxH 690T hematology analyzer. Fig. 9BAs shown, there is a linear relationship between the concentration of bovine HGB and the concentration of human HGB, thereby allowing accurate reproduction of any specific hemoglobin-related disease, as discussed in the above paragraph. Example 5: Hemolysis of hydrogel particles

[0176] Chemically or physically labile cross-linkers or cleavable polymer chains can be incorporated into the hydrogel polymer matrix to mimic salt- or surfactant-induced hemolysis of erythrocytes.

[0177] In this example, hydrogel particles are synthesized by copolymerizing acrylamide with bis(2-methacryloyl)oxyethyl disulfide, which acts as a redox-cleavable crosslinker. The scattering properties and Coulter volume of the hydrogel particles are adjusted to match human erythrocytes. When exposed to 5wt% 1,4-dithiothreitol (DTT) or β-mercaptoethanol (BME) (e.g., 2-mercaptoethanol) solution, the disulfide is reduced to a thiol and the crosslinker is cleaved.

[0178] As a result, the hydrogel matrix breaks into water-soluble individual polyacrylamide chains and dissolves. After dissolution, the hydrogel particles are no longer detected by flow cytometry or the Coulter principle. Therefore, the dissolved particles do not interfere with the measurement of other more stable particles (such as leukocytes or their mimics) that may be in the same suspension. Example 6: Compatibility test of hydrogel lysis buffer with whole blood measurement

[0179] This example was designed to test whether the hydrogel lysis buffer would affect the measurement of whole blood cell samples by a hematology analyzer. Different concentrations of DTT (10 mM, 100 mM, and 300 mM) were added to whole blood cell samples containing the red blood cell control composition disclosed herein. The mixture was immediately placed in a Beckman Coulter DxH 690T hematology analyzer to measure the characteristics of the whole blood sample. Fig.10 As shown, DTT did not affect the measurement. Approximately 2 minutes after the addition of DTT, the red blood cell control composition was lysed and diffusion was arrested.

[0180] Another reducing agent, TCEP, was also tested at a concentration of 25 mM. The results showed that TCEP also did not affect the measurement (data not shown). ********

[0181] All documents, patents, patent applications, publications, product specifications, and protocols cited throughout this application are hereby incorporated by reference in their entirety for all purposes.

[0182] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best ways known to the inventors to make and use the invention of the present disclosure. Modifications and variations of the above embodiments of the present disclosure are possible without departing from the spirit of the present invention, as will be appreciated by those skilled in the art in light of the above teachings. Therefore, it should be understood that within the scope of the claims and their equivalents, the invention of the present disclosure may be practiced in a manner other than that specifically described.

Claims

1. A red blood cell control composition, comprising: (i) one or more populations of cleavable hydrogel particles having an impedance substantially similar to the impedance of a human red blood cell having an average diameter; and (ii) a population of hemoglobin molecules or a population of dye molecules having an absorbance substantially similar to that of hemoglobin; wherein (i) and / or (ii) are present in an amount corresponding to a normal blood sample or a disease state, or (i) and (ii) are present in a ratio corresponding to a normal blood sample or a disease state.

2. The red blood cell control composition of claim 1, wherein at least one hemoglobin molecule or dye molecule is encapsulated within each cleavable hydrogel.

3. The red blood cell control composition of claim 1, wherein at least one hemoglobin molecule or dye molecule is attached to each cleavable hydrogel.

4. The red blood cell control composition of claim 3, wherein the hemoglobin molecule or the dye molecule is covalently attached to the cleavable hydrogel.

5. The red blood cell control composition of claim 3, wherein the hemoglobin molecule or the dye molecule is non-covalently attached to the cleavable hydrogel.

6. The red blood cell control composition of claim 1, wherein at least one hemoglobin molecule or dye molecule is not encapsulated within or attached to each cleavable hydrogel.

7. The red blood cell control composition of claim 6, wherein the composition is a solution, and the population of hemoglobin molecules or the population of dye molecules are independently suspended or dissolved in the solution, and the one or more populations of cleavable hydrogel particles are independently suspended in the solution.

8. The red blood cell control composition of any one of claims 1-7, wherein the cleavable hydrogel comprises polymerized monomers and co-monomers.

9. The red blood cell control composition of claim 8, wherein the comonomer is a difunctional monomer.

10. The red blood cell control composition of claim 9, wherein the bifunctional monomer comprises a bond that can be cleaved by a lysis buffer.

11. The red blood cell control composition of claim 9 or 10, wherein the bifunctional monomer comprises a disulfide bond.

12. The red blood cell control composition according to any one of claims 9 to 11, wherein the bifunctional monomer is N,N'-bis(acryloyl)cystamine, bis(2-methacryloyl)oxyethyl disulfide, allyl disulfide, polyethylene glycol (PEG) N-hydroxysuccinimide (NHS) ester disulfide, acryloyl-PEG-disulfide-PEG-acryloyl or 3-(2-pyridyldithio)propionic acid succinimidyl ester.

13. The red blood cell control composition according to any one of claims 10-12, wherein the lysis buffer comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), β-mercaptoethanol (BME) or glutathione (GSH).

14. The red blood cell control composition of claim 9 or 10, wherein the bifunctional monomer comprises an acid-labile bond.

15. The red blood cell control composition according to any one of claims 9, 10 and 14, wherein the bifunctional monomer is diisopropylbenzyl alcohol dimethacrylate, diisopropylbenzyl alcohol diacrylate, 2,5-dimethyl-2,5-hexanediol dimethacrylate, acylhydrazone or 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane.

16. The red blood cell control composition of any one of claims 10, 14, and 15, wherein the lysis buffer comprises formic acid.

17. The red blood cell control composition of any one of claims 1-16, further comprising one or more additional molecules encapsulated in each cleavable hydrogel.

18. The red blood cell control composition of claim 17, wherein the one or more additional molecules are one or more polynucleotides, one or more polypeptides, a complex of one or more polynucleotides and one or more polypeptides, or a combination thereof.

19. The red blood cell control composition of any one of claims 1-18, wherein the one or more cleavable hydrogel particle populations have an average diameter ranging from about 1 μm to about 20 μm.

20. The red blood cell control composition of any one of claims 1-18, wherein the one or more cleavable hydrogel particle populations have an average diameter ranging from about 1 μm to about 15 μm.

21. The red blood cell control composition of any one of claims 1-18, wherein the one or more cleavable hydrogel particle populations have an average diameter ranging from about 2 μm to about 15 μm.

22. The red blood cell control composition of any one of claims 1-18, wherein the one or more cleavable hydrogel particle populations have an average diameter ranging from about 5 μm to about 15 μm.

23. The red blood cell control composition of any one of claims 1-18, wherein the one or more cleavable hydrogel particle populations have an average diameter ranging from about 5 μm to about 10 μm.

24. The red blood cell control composition of any one of claims 1-18, wherein the one or more cleavable hydrogel particle populations have an average diameter of about 7 μm to about 9 μm.

25. The red blood cell control composition of any one of claims 1-24, wherein the composition has a range of about 4.2 x 10 RBCs when measured using impedance-based cytometry. 9 / mL to about 5.9x10 9 / mL.

26. The red blood cell control composition of any one of claims 1-24, wherein the composition has less than 4.2 x 10 RBCs when measured using impedance-based cytometry. 9 / mL.

27. The red blood cell control composition of any one of claims 1-24, wherein the composition has an RBC count greater than 5.9 x 10 9 / mL.

28. The red blood cell control composition of any one of claims 1-27, wherein the composition has an HGB ranging from about 12 g / dL to about 17 g / dL when measured by absorbance.

29. The red blood cell control composition of any one of claims 1-27, wherein the composition has a HGB of less than 12 g / dL when measured by absorbance.

30. The red blood cell control composition of any one of claims 1-27, wherein the one or more populations of cleavable hydrogel particles have a HGB greater than 17 g / dL when measured by absorbance.

31. The red blood cell control composition of any one of claims 1-30, wherein the MCV of the composition ranges from about 80 fL to about 100 fL when measured using impedance-based cytometry.

32. The red blood cell control composition of any one of claims 1-30, wherein the MCV of the composition is less than 80 fL when measured using impedance-based cytometry.

33. The red blood cell control composition of any one of claims 1-30, wherein the MCV of the composition is greater than 100 fL when measured using impedance-based cytometry.

34. The red blood cell control composition of any one of claims 1-33, wherein the MCH of the one or more populations of cleavable hydrogel particles ranges from about 28 pg to about 32 pg when measured using impedance-based cytometry.

35. The red blood cell control composition of any one of claims 1-33, wherein the MCH of the composition is less than 28 pg when measured using impedance-based cytometry.

36. The red blood cell control composition of any one of claims 1-33, wherein the MCH of the composition is greater than 32 pg when measured using impedance-based cytometry.

37. The red blood cell control composition of any one of claims 1-36, wherein the RDW of the composition ranges from about 11% to about 16% when measured using impedance-based cytometry.

38. The red blood cell control composition of any one of claims 1-36, wherein the RDW of the composition is less than about 11% when measured using impedance-based cytometry.

39. The red blood cell control composition of any one of claims 1-36, wherein the RDW of the composition is greater than about 16% when measured using impedance-based cytometry.

40. The red blood cell control composition of any one of claims 1-39, wherein the composition has less than about 100 / 100 μL of NRBCs when measured using impedance-based cytometry.

41. The red blood cell control composition of any one of claims 1-39, wherein the composition has greater than about 100 / 100 μL of NRBCs when measured using impedance-based cytometry.

42. The red blood cell control composition of any one of claims 1-41, wherein the peak excitation wavelength of the dye molecule having a substantially similar absorbance to hemoglobin ranges from about 600 nm to about 750 nm.

43. The red blood cell control composition of any one of claims 1-42, wherein the peak emission wavelength of the dye molecule having a substantially similar absorbance to hemoglobin ranges from about 625 nm to about 775 nm.

44. The red blood cell control composition of any one of claims 1-43, further comprising a population of synthetic white blood cells.

45. The red blood cell control composition of claim 44, wherein the synthetic leukocytes comprise one or more populations of non-cleavable hydrogel particles.

46. ​​The red blood cell control composition of claim 45, wherein the non-cleavable hydrogel comprises a polymer comprising a polymerized monomer and a co-monomer.

47. The red blood cell control composition of any one of claims 44-46, wherein the synthetic leukocytes have an impedance substantially similar to the impedance of human leukocytes having an average diameter.

48. The red blood cell control composition of any one of claims 44-47, wherein the synthetic leukocytes are synthetic monocytes, granulocytes, or lymphocytes.

49. The red blood cell control composition of claim 48, wherein the granulocytes are neutrophils, eosinophils, or basophils.

50. The red blood cell control composition of claim 48, wherein the lymphocytes are T cells or B cells.

51. A synthetic whole blood sample control, the synthetic whole blood sample control comprising: (i) one or more populations of cleavable hydrogel particles having an impedance substantially similar to the impedance of a human red blood cell having an average diameter; (ii) a population of hemoglobin molecules or a population of dye molecules having an absorbance substantially similar to that of hemoglobin; and (iii) one or more populations of non-cleavable hydrogel particles having an impedance substantially similar to the impedance of a human leukocyte having an average diameter.

52. A method for evaluating a red blood cell sample using a hematology analyzer, the method comprising: (a) measuring one or more properties of the red blood cell control composition according to any one of claims 1-50; (b) measuring one or more characteristics of the red blood cell sample; and (c) comparing the measurement results of the red blood cell sample with the measurement results of the control sample.

53. The method of claim 52, wherein the one or more cleavable hydrogel particle populations are (a) an average diameter of about 7 μm to about 9 μm; (b) The range of RBC is about 4.2 x 10 9 / mL to about 5.9x 10 9 / mL; (c) HGB ranging from about 12 g / dL to about 17 g / dL; (d) MCV ranges from about 80 fL to about 100 fL; (e) MCH ranges from about 28 pg to about 32 pg; (f) RDW in the range of about 11% to about 16%; and / or (g) NRBC is less than about 100 / 100 μL.

54. The method of claim 52 or 53, wherein a deviation in the measurement result of the red blood cell sample from the measurement result of the red blood cell control composition is indicative of a condition or disease.

55. The method of claim 52, wherein the one or more cleavable hydrogel particle populations are (a) The average diameter is less than 7 μm; (b) average diameter higher than 9 μm; (c) RBC less than 4.2 x 10 9 / mL; (d) RBC is higher than 5.9 x 10 9 / mL; (e) HGB less than 12 g / dL; (f) HGB higher than 17 g / dL; (g) MCV less than 80 fL; (h) MCV higher than 100 fL; (i) MCH less than 28 pg; (j) MCH higher than 32 pg; (k) RDW less than 11%; (l) RDW greater than 16%; and / or (g) NRBC is higher than 100 / 100 μL.

56. The method of claim 55, wherein a substantial similarity between the measurement results of the red blood cell sample and the measurement results of the control sample is indicative of a condition or disease.

57. The method of any one of claims 52-56, wherein the measuring uses a pore-based technique, an image-based technique, and / or a waveform-based technique.

58. The method of any one of claims 52-57, wherein the hematology analyzer is an optical-based cytometer.

59. The method of any one of claims 52-57, wherein the hematology analyzer is a resistance-based cytometer.

60. The method of any one of claims 52-59, wherein the condition or disease is elevated NRBC, bone marrow failure, anemia, erythrocyte enzyme deficiency, erythrocyte membrane disorder, hemoglobinopathy, polycythemia, hemochromatosis, hyperhemoglobin, hypohemoglobin, polycythemia, anisocytosis, macrocytosis, microcytosis, or leukemia.

61. The red blood cell control composition of claim 46, wherein the monomer is acetic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxy methacrylate, ethyl acrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glyceryl methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate acrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N-phenylmethacrylamide, N-benzylacrylamide, N-benzylmethacrylamide, N,N-dibenzylacrylamide, N,N-dibenzylmethacrylamide, N-diphenyl Methacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-tritylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylacrylamide, N,N-phenylphenethylacrylamide, N-dibenzhydrylmethacrylamide, N-(4-methylphenyl)methylmethacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-tritylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethacrylamide, N,N'-phenylphenethyl methacrylamide, N-vinyl carbazole, 4-vinyl pyridine, 2-vinyl pyridine methacrylamide, N-alkyl acrylamide, N-alkyl methacrylamide, N,N-dialkyl acrylamide, N-[(dialkylamino)alkyl] acrylamide, N-[(dialkylamino)alkyl] methacrylamide, (dialkylamino)alkyl acrylate or (dialkylamino)alkyl methacrylate.

62. The red blood cell control composition of claim 46, wherein the comonomer is allylamine, allyl acrylate, allyl methacrylate, allyl alcohol, allyl isothiocyanate, allyl chloride, allyl maleimide, or bisacrylamide.

63. The population of hydrogel particles of claim 62, wherein the bisacrylamide is N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, N,N'-ethylenebisacrylamide, N,N'-ethylenebismethacrylamide, N,N'-propylenebisacrylamide, or N,N'-(1,2-dihydroxyethylene)bisacrylamide.

64. The red blood cell control composition of claim 8, wherein the monomer is acetic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxy methacrylate. Ethyl acrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glyceryl methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methyl Acrylates, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenylacrylamide, N-phenylmethacrylamide, N-benzylacrylamide, N-benzylmethacrylamide, N,N-dibenzylacrylamide, N,N-dibenzylmethacrylamide, N-diphenyl Methacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-tritylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylacrylamide, N,N-phenylphenethylacrylamide, N-dibenzhydrylmethacrylamide, N-(4-methylphenyl)methylmethacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-tritylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethacrylamide, N,N'-phenylphenethyl methacrylamide, N-vinyl carbazole, 4-vinyl pyridine, 2-vinyl pyridine methacrylamide, N-alkyl acrylamide, N-alkyl methacrylamide, N,N-dialkyl acrylamide, N-[(dialkylamino)alkyl] acrylamide, N-[(dialkylamino)alkyl] methacrylamide, (dialkylamino)alkyl acrylate or (dialkylamino)alkyl methacrylate.

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