Particles produced as red blood cell mimics for hematology and compositions containing them

Abstract

An erythrocyte control composition comprising one or more populations of dissolvable hydrogel particles having an impedance substantially similar to that of human erythrocytes of average diameter, and a population of hemoglobin molecules or a population of dye molecules having an absorbance substantially similar to hemoglobin, wherein (i) and / or (ii) are present in an amount corresponding to a normal blood sample or a medical condition, or (i) and (ii) are present in a ratio corresponding to a normal blood sample or a medical condition, is provided.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority and the benefit thereof to U.S. Provisional Application No. 63 / 338,719, filed May 5, 2022, the entire content of which is incorporated herein by reference.

[0002] Field The present disclosure generally relates to erythrocyte control compositions and their use for evaluating erythrocyte samples. The present disclosure also relates to synthetic whole blood sample controls that include both erythrocyte control compositions and leukocyte control compositions.

Background Art

[0003] Background of the Invention Variations in the morphological and physiological characteristics of erythrocytes in a patient's blood provide useful information regarding the pathological states of many specific types of erythrocyte disorders or anemia. In the diagnosis of such disorders, measuring the mean cellular hemoglobin concentration (MCHC) and the mean corpuscular volume (MCV) can provide useful insights into the patient's condition. Such information can be used together with microscopic evaluation by a trained hematologist of the size, shape, and color distribution of erythrocytes in stained blood smears, as well as other biochemical tests. Variations in the refractive index of individual erythrocytes are highly correlated with their hemoglobin concentration, and this information, combined with size measurements, can provide diagnostic values. For example, in microcytic anemia, the size of erythrocytes is significantly reduced, and thus the MCV is also significantly reduced, but the optical density (related to the refractive index) and MCHC increase. In megaloblastic anemia, both the size (macrocytes) and MCHC increase.

[0004] Fluctuations in the characteristics or quantity of reticulocytes in a patient's blood may indicate erythrocyte production by the patient's bone marrow and can assist in diagnosing various conditions, such as anemia or bone marrow failure. Detection of fluctuations in the characteristics or quantity of platelets (thrombocytes), which assist the coagulation system, can assist in diagnosing thrombocythemia, reactive thrombocytosis, thrombocytopenia, and platelet dysfunction.

[0005] Considering the above, the ability to identify fluctuations in the morphological and physiological characteristics of erythrocytes, reticulocytes, platelets, and other blood components is very important for the evaluation of patients and successful outcomes.

[0006] Flow cytometry is a technique that enables the rapid separation, counting, and characterization of individual cells, such as erythrocytes, and is routinely used in clinical and laboratory settings for various applications. Optical-based flow cytometry relies on directing a light beam at a liquid stream focused by hydrodynamics. Next, at the point where the stream passes through the light beam, one detector is aligned with the light beam (forward scatter or FSC) and several detectors are perpendicular to the light beam (side scatter or SSC), defining several detectors. FSC correlates with cell volume, and SSC depends on the internal complexity of the particle (e.g., nuclear shape, amount and type of cytoplasmic granules, or membrane roughness). As a result of these correlations, different specific cell types exhibit different FSC and SSC, allowing the cell types to be distinguished.

[0007] However, optical-based flow cytometry is an expensive technology that requires procurement of costly reagents. As a result, more common and simpler techniques, such as impedance-based methods, are deployed as the basis for primary measurements in clinical settings. The electrical impedance-based method is rooted in the Coulter principle. During such an electrical impedance-based method, whole blood can be passed between two electrodes through an aperture that is narrow enough so that only a single cell can pass through at a time. The impedance between the electrodes changes as a cell passes between the electrodes and is proportional to the cell volume, allowing the cells to be counted and evaluated (e.g., volume measurement). Impedance-based techniques can calculate the total blood cell count, determine a three-part white blood cell fraction, and distinguish, for example, red blood cells, white blood cells, and platelets, although they cannot determine the types of granulocytes of similar size. As a result of its robustness and applicability to general clinical problems, the electrical impedance-based technique is widely practiced in the clinic and serves as the technical basis for almost all commercially available blood analyzers, such as the DxH900, DxH690 analyzers (Beckman Coulter, Inc.), and the XN-1000, XN-L analyzers (Sysmex, Inc.).

[0008] However, the ability to identify and measure specific cell types, such as red blood cells, reticulocytes, and platelets, relies on proper calibration of the measuring instrument. In the case of a blood analyzer, for example, calibration has relied on the use of purified cells of the cell type of interest. Obtaining these purified cells can require expensive and laborious procedures that tend to be subject to batch-to-batch variation. These purified cells may also be augmented with cells obtained from endangered species, such as alligators and sharks. Therefore, there is a need in the art for synthetic compositions having adjustable optical and electrical properties that can mimic red blood cells, reticulocytes, and platelets in a blood analyzer. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0009] Summary of the Invention In some embodiments, the present disclosure relates to an erythrocyte control composition comprising a population of created particles (e.g., hydrogel) and a population of hemoglobin or hemoglobin-like molecules. In some embodiments, hemoglobin-like molecules include, for example, heme, dyes (e.g., having an absorbance substantially similar to hemoglobin), or oxygen-carrying molecules, or oxygen-transporting molecules. In certain embodiments, the hemoglobin-like molecule is a dye. Such hemoglobin-like molecules can produce a spectral response substantially similar to the spectral response of hemoglobin. In some embodiments, each created particle (e.g., hydrogel) comprises at least one hemoglobin or hemoglobin-like molecule encapsulated within the hydrogel or conjugated to the hydrogel. In some embodiments, the population of hemoglobin or hemoglobin-like molecules is independent of the created particles (hydrogel) (not conjugated and not encapsulated). In some embodiments, the created particles (e.g., hydrogel) can have at least one representative characteristic substantially similar to a corresponding characteristic of erythrocytes. In some embodiments, the at least one representative characteristic is one or more of an optical characteristic and a morphological characteristic. In some embodiments, the morphological characteristic is one or more of diameter and volume. In some embodiments, one or more hemoglobin or hemoglobin-like molecules are encapsulated within the created particles, or one or more hemoglobin or hemoglobin-like molecules are bound to the surface of the created particles (e.g., hydrogel). In some embodiments, the created particles (e.g., hydrogel) are degradable. In some embodiments, the created particles (e.g., hydrogel) are soluble by a blood lysis buffer. In some embodiments, the at least one representative characteristic is determined by an aperture-based technique, an image-based technique, and / or a waveform-based technique. In some embodiments, each created particle has an average diameter between about 1 μm and about 20 μm.In some embodiments, the population of particles produced further comprises a plurality of subpopulations of the particles produced, each subpopulation of the particles produced has at least one representative feature, and the at least one representative feature includes one or more of an optical feature and a morphological feature, and the morphological feature includes at least one of a diameter and a volume. In some embodiments, the hemoglobin molecule is a hemoglobin molecule of a human, mouse, cow, sheep, bird, dog, cat, pig, or plant.

[0010] In some embodiments, the present disclosure provides an erythrocyte control composition comprising (i) one or more populations of lytic (e.g., soluble in the presence of a lysis buffer) hydrogel particles having an impedance substantially similar to that of human erythrocytes of 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 medical condition, or (i) and (ii) are present in a ratio corresponding to a normal blood sample or a medical condition.

[0011] In some embodiments, a population of hemoglobin molecules or a population of dye molecules is encapsulated within one or more populations of soluble hydrogels. In some embodiments, at least one hemoglobin molecule or dye molecule is encapsulated within each soluble hydrogel. In some embodiments, a population of hemoglobin molecules or a population of dye molecules is bound to one or more populations of soluble hydrogels. In some embodiments, at least one hemoglobin molecule or dye molecule is bound to each soluble hydrogel. In some embodiments, one hemoglobin molecule or dye molecule is covalently bound to each soluble hydrogel. In some embodiments, at least one hemoglobin molecule or dye molecule is non-covalently bound to each soluble hydrogel (e.g., via a biotin / streptavidin interaction). In some embodiments, at least one hemoglobin molecule or dye molecule is neither encapsulated within nor bound to each soluble hydrogel. In some embodiments, the composition is a solution, a population of hemoglobin molecules or dye molecules is suspended in the solution, and one or more populations of soluble hydrogel particles are suspended in the solution. BRIEF DESCRIPTION OF THE DRAWINGS

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DETAILED DESCRIPTION OF THE INVENTION

[0021] DETAILED DESCRIPTION OF THE INVENTION This specification describes an erythrocyte control composition. The erythrocyte control composition is created to provide absorbance and impedance characteristics that are substantially similar to those of a human whole blood sample when measured using a blood analyzer. To achieve this, the composition includes (i) one or more populations of soluble hydrogel particles having an impedance substantially similar to that of human erythrocytes of average diameter, and (ii) a population of hemoglobin molecules or a population of dye molecules having an absorbance substantially similar to hemoglobin. One or more populations of soluble hydrogel particles having an impedance substantially similar to that of human erythrocytes of average diameter can be interchangeably referred to herein as erythrocyte mimics or RBC mimics.

[0022] Advantageously, the erythrocyte control composition can be processed by a blood analyzer in the same manner as a human whole blood sample without interfering with the blood analyzer, and thus can be used as a control for the blood analyzer. For example, in some embodiments, the size (e.g., based on forward scatter), complexity (e.g., based on side scatter), and number (e.g., based on impedance) of the synthetic erythrocytes can be measured. In some embodiments, the erythrocyte control composition can be combined with synthetic leukocytes (e.g., as described in U.S. Patent No. 10,753,846, which is hereby incorporated by reference in its entirety) to form a synthetic whole blood sample. The synthetic whole blood sample can then be introduced into a blood analyzer, and the size, complexity, and number of both the synthetic erythrocytes and leukocytes can be measured. In some embodiments, the synthetic erythrocytes of the synthetic whole blood sample can be lysed so that the size, complexity, and number of the synthetic leukocytes can be measured. In some embodiments, absorbance can be measured to quantify the amount of hemoglobin or dye.

[0023] The red blood cell control composition can be adjusted to match the impedance and absorbance of the target red blood cell composition. For example, in some 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, or a blood sample from a subject having any other disease characterized by anisocytosis or abnormal hemoglobin levels or characteristics of red blood cells (e.g., size, complexity, and / or number). In some embodiments, the subject is a human subject.

[0024] Definitions Unless otherwise defined herein, technical and scientific terms used in this description have the meanings commonly understood by one of ordinary skill in the art. For the purposes of interpreting this specification, the following explanations of terms apply, and whenever appropriate, terms used in the singular include the plural and vice versa, unless the context clearly indicates otherwise. If any explanation of a term described conflicts with any document incorporated herein by reference, the explanation of the following terms 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 otherwise clearly indicated by the context in which they are used.

[0026] "At least one" and "one or more" are used interchangeably to mean that an article can include one or more of the recited elements.

[0027] It should be understood that all numerical values representing amounts, ratios, and numerical characteristics of components, reaction conditions, etc., used in this specification and the claims, are intended to be modified in all instances by the term "about" unless otherwise specified.

[0028] As used herein, the term "about", when referring to a numerical value or range of numerical values, is understood to mean the recited numerical value and numerical values within + / - 10% thereof, or a numerical value 10% less than the recited lower limit value and a numerical value 10% greater than the recited upper limit value, with respect to the values recited for a range.

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

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

[0031] As used herein, the term "comprise" and its grammatical equivalents identify the presence of the described 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 groups thereof.

[0032] As used herein, the term "normal" describes a subject that is healthy and has no disease or condition, or a biological sample from such a subject.

[0033] Cytometer As shown in FIG. 1A, for an optical-based cytometer, side scatter provides a general measure of cell complexity, while forward scatter provides a measure of particle size. The two most important passive optical measurements used in optical-based flow cytometry are FSC (forward scatter) and SSC (side scatter), which measure the size and complexity of the target, respectively. Currently, due to their limitations with polystyrene, users have to rely on purified cell lines to calibrate fluorescence intensity, laser-to-laser delay, sort delay, size, and cell complexity for experiments. This is a cumbersome and labor-intensive process that significantly increases the cost of the verification and research pipeline by optical-based flow cytometry. More importantly, these calibration cell lines introduce biological variability and cause differences in data interpretation.

[0034] In electrical impedance-based cytometry based on the Coulter principle (shown in FIG. 1B), two chambers separated by a microchannel are utilized, and each of the two chambers contains an electrolyte solution. When a particle or cell containing fluid is drawn through the microchannel between the two separate chambers, each particle causes a short-term change in the electrical resistance of the liquid. Further information regarding impedance-based cytometry can be found at www.beckman.com / resources / technologies / coulter-principle / coulter-principle-short-course-chapter-1, the entirety of which is incorporated herein by reference for all purposes.

[0035] In other words, particles drawn through the microchannel simultaneously with the current cause a change in impedance proportional to the volume of the particles across the microchannel. This impedance pulse results from the displacement of the electrolyte caused by the particles. Cells, which are particles with low conductivity, change the effective cross-sectional area of the conductive microchannel. When these particles are less conductive than the surrounding liquid medium, the electrical resistance across the channel increases, causing a short-term decrease in the current passing through the microchannel. By monitoring such current pulses, the number of particles in a given volume of fluid can be counted. Furthermore, the magnitude of the current change is related to the particle size, enabling measurement of the particle size distribution, which can correlate with the particle mobility, surface charge, and concentration.

[0036] For any flow cytometry technique, calibration is very important for accurate performance. The disclosed created particles exhibit regulated properties and are suitable for use as calibration reagents for various mammalian or bacterial cell types, including red blood cells.

[0037] In some embodiments, a composition is provided that includes a plurality of created particles, each of the plurality of created particles having one or more characteristics that are substantially similar to one or more characteristics of a target cell. In some embodiments, each of the created particles, i.e., each of the plurality of individual created particles, is synthesized by polymerizing one or more monomers, i.e., to form a homopolymer or a copolymer. As further discussed below, the use of bifunctional monomers enables further derivatization of hydrogels using, for example, fluorescent dyes, encapsulated proteins, cell surface markers or their epitope-binding fragments, or combinations thereof. An example of adjusting particle parameters created to meet / match the measurement criteria of a desired cell subpopulation is provided in FIG. 4. Methods for adjusting the characteristics of the created particles are described herein. Embodiments for how to adjust the forward scatter, side scatter, and surface characteristics of the created particles are provided in FIGS. 5 and 6. The ability to adjust various parameters including the components and their concentrations of the created particles enables the particles to be adjusted to mimic, as an example, the characteristics of red blood cells. For example, in an attempt to mimic the Coulter volume (related to impedance) of red blood cells, the actual volume and / or porosity of the particles can be adjusted. It should be understood that the Coulter volume of the particles (the apparent volume measured by the Coulter principle) depends on the amount of electrolyte replaced by the particles. For cells with an intact membrane, their Coulter volume is similar to the actual volume. For the particles described herein, the amount of electrolyte 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 the actual size of the particles or independently of the actual size of the particles. In the example of red blood cells, red blood cell-mimicking particles with diameters varying between 5 μm and 10 μm can be synthesized by adjusting conditions such as microfluidic pressure or microfluidic device design.The Coulter volume of these erythrocyte-mimicking particles can be designed to be higher than, lower than, or equal to that of actual erythrocytes, or normal and / or abnormal subpopulations thereof, as required.

[0038] Optical properties As provided above, in one aspect, the present disclosure provides individually created particles each having one or more properties that are substantially similar to one or more properties of target cells, such as erythrocytes, reticulocytes, or platelets. In some embodiments, the one or more properties can be optical properties. In some embodiments where the one or more properties include optical properties, the optical properties can include a side scatter profile, a forward scatter profile, an angular light scatter profile, or a secondary marker profile, such as a fluorescence marker profile, an absorption profile, a fluorescence profile, or an emission profile.

[0039] In some embodiments, the optical properties are substantially similar to the absorption properties of erythrocytes. The absorbance by erythrocytes rapidly decreases above about 600 nm and varies slightly depending on whether the erythrocytes are oxygen-bound. The spectral waveform of hemoglobin is provided at omic.org / spectra / hemoglobin. Pigments can also be used to mimic the absorbance of erythrocytes.

[0040] In some embodiments, the dye having an absorbance substantially similar to hemoglobin is a red dye. In some embodiments, the dye having an absorbance substantially similar to hemoglobin has a peak excitation wavelength (nm) of from about 600 to about 750, such as 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, 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 therebetween).

[0041] In some embodiments, the dye having an absorbance substantially similar to hemoglobin is a red dye. In some embodiments, the dye having an absorbance substantially similar to hemoglobin has a peak emission wavelength (nm) of about 625 to about 775, such as 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, 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 therebetween).

[0042] In some embodiments, the dye is Allura Red AC (also known as Red No. 40). In some embodiments, the dye is India Ink. In some embodiments, the dye is EPolight 2717. In some embodiments, the particles produced are derivatized with one or more of the following fluorescent dyes: 6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein succinimidyl ester; 5-(and-6)-carboxy eosin; 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; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2’,7’-difluorofluorescein; eosin-5-isothiocyanate; erythrosin 5-isothiocyanate; 6-(fluorescein-5-carboxamide)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-carboxamide)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; OregonGreen® 488 carboxylic acid, or succinimidyl ester; Oregon Green® 488 isothiocyanate; Oregon Green® 488-X succinimidyl ester; Oregon Green® 500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon Green® 514 carboxylic acid; Oregon Green® 514 carboxylic acid or succinimidyl ester; RhodamineGreen™ carboxylic acid, succinimidyl ester or hydrochloride;Rhodamine Green(trademark) carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green(trademark)-X succinimidyl ester or hydrochloride; RhodolGreen(trademark) carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl) sulfon rhodamine or di(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’-tetrabromosulfonfluorescein succinimidyl ester or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimidyl ester; 6-carboxytetramethylrhodamine succinimidyl ester; 5-(and-6)-carboxytetramethylrhodamine succinimidyl ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester; 6-carboxy-X rhodamine succinimidyl ester; 5-(and-6)-carboxy-X rhodamine succinimidyl ester; 5-carboxy-X-rhodamine triethylammonium salt; Lissamine(trademark) rhodamine B sulfonyl chloride; malachite green; isothiocyanate; NANOGOLD(registered trademark) mono(sulfosuccinimidyl ester); QSY(registered trademark) 21 carboxylic acid or succinimidyl ester; QSY(registered trademark) 7 carboxylic acid or succinimidyl ester; Rhodamine Red(trademark)-X succinimidyl ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamide) hexanoic acid; succinimidyl ester;Tetramethylrhodamine-5-isothiocyanate; Tetramethylrhodamine-6-isothiocyanate; Tetramethylrhodamine-5-(and -6)-isothiocyanate; Texas Red® Sulfonyl; Texas Red® Sulfonyl Chloride; Texas Red®-X STP Ester or Sodium Salt; Texas Red®-X Succinimidyl Ester; Texas Red®-X Succinimidyl Ester; and X-Rhodamine-5-(and -6) Isothiocyanate, including but not limited to BODIPY® FL, BODIPY® TMR STP Ester, BODIPY® TR-X STP Ester, BODIPY® 630 / 650-X STP Ester, BODIPY® 650 / 665-X STP Ester, BODIPY® dyes commercially available from Invitrogen; 6-Dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic Acid Succinimidyl Ester; 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic Acid; 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic Acid; 4,4-Difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-pentanoic Acid Succinimidyl Ester; 4,4-Difluoro-5,7-dimethyl(dimefhyl)-4-bora-3a,4a-diaza-s-indacene-3propionic Acid; 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic Acid Succinimidyl Ester; 4,4Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3propionic Acid; Sulfosuccinimidyl Ester or Sodium Salt; 6-((4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3propionyl)amino)hexanoic Acid; 6-((4,4-Difluoro-5,7dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic Acid or Succinimidyl Ester;N-(4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-propionyl)cysteic acid, succinimidyl 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-s-indacene-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-bora 3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; succinimidyl ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacen-3-propionyl)amino)hexanoic acid or succinimidyl ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacen-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester;6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacen-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester, including but not limited to Alexa Fluor® 350 carboxylic acid, Alexa Fluor® 430 carboxylic acid, Alexa Fluor® 488 carboxylic acid, Alexa Fluor® 532 carboxylic acid, Alexa Fluor® 546 carboxylic acid, Alexa Fluor® 555 carboxylic acid, Alexa Fluor® 568 carboxylic acid, Alexa Fluor® 594 carboxylic acid, Alexa Fluor® 633 carboxylic acid, Alexa Fluor® 647 carboxylic acid, Alexa Fluor® 660 carboxylic acid, and Alexa Fluor® 680 carboxylic acid, Alexa fluor dyes commercially available from Invitrogen, and including but not limited to Cy3 NHS ester, Cy5 NHS ester, Cy5.5 NHS ester, and Cy7 NHS ester, cyanine dyes commercially available from Amersham-Pharmacia Biotech.;

[0043] In some embodiments, the created particles are "rainbow particles" containing a mixture of multiple types of fluorophores, such as 4 types of fluorophores, 5 types of fluorophores, 6 types of fluorophores, 7 types of fluorophores, or 8 types of fluorophores. In this regard, the user selects at which wavelength to excite the particles depending on the fluorophore being examined. Rainbow particles are commercially available, for example, from BD Biosciences (catalog numbers 556298 (intermediate region FL1 fluorescence), 556286 (6 colors, 3.0 - 3.4 μm), 556288 (6 colors, 6.0 - 6.4 μm), 559123 (8 colors)) and Spherotech in various diameters (e.g., catalog numbers RCP20 - 5 (4 colors), RCP - 30 - 5 (6 peaks), RCP - 30 - 5A (8 peaks)).

[0044] Hemoglobin-like molecules can be used to mimic the absorbance of red blood cells. For example, heme is a precursor of hemoglobin. Also, hemoglobin can be derived from human, mouse, bovine, ovine, avian, canine, feline, porcine, or plant sources. Hemoglobin from leguminous plants can be derived from soybean plants and can be produced by bioengineered yeast. Hemoglobin variants include Gower 1, Gower 2, hemoglobin Portland1, hemoglobin PortlandII, hemoglobin F, hemoglobin A, hemoglobin A 2 , hemoglobin F, hemoglobin D-Punjab, hemoglobin H, hemoglobin Bart, hemoglobin S, hemoglobin C, hemoglobin E, hemoglobin AS, hemoglobin SC, and hemoglobin Hopkins-2. Hemoglobin-like molecules include myoglobin, hemocyanin, hemerythrin, chlorocruorin, vanabin, erythrocruorin, pinnaglobin, leghemoglobin, and coboglobin.

[0045] One or more of the surfaces of the particles can be functionalized, for example, to mimic one or more optical properties of target cells or labeled target cells. The functionalized hydrogel particles can also contain encapsulated beads or substances, such as biomolecules, as described above. In some embodiments, one or more hydrogel particles are functionalized with one or more fluorescent dyes, one or more cell surface markers (or their epitope binding regions), or combinations thereof. In some embodiments, the hydrogel particles are formed by polymerizing at least one bifunctional monomer, and after formation, the hydrogel particles contain one or more functional groups that can be used for further attachment of cell surface markers, epitope binding regions of cell surface markers, fluorescent dyes, or combinations thereof. The free functional groups are, in some embodiments, amine groups, carboxyl groups, hydroxy groups, or combinations thereof. It should be understood that multiple bifunctional monomers can be used depending on the desired functionalization, for example, using different chemicals and different molecules to functionalize the particles.

[0046] The resulting particles can be functionalized with any of the fluorescent dyes known in the art, including those listed in MolecularProbes® Handbook - A Guide to Fluorescent Probes and Labeling Technologies, which is hereby incorporated by reference in its entirety for all purposes. The functionalization can be mediated, as discussed above, by a compound containing a free amine group, such as allylamine, that can be incorporated into the bifunctional monomer used to form the hydrogel.

[0047] Non-limiting examples of known fluorescent dyes that can be used to functionalize the surface of the particles described in this specification include 6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein succinimidyl ester; 5-(and -6)-carboxy eosin; 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; 5-(and -6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl) aminofluorescein; 2’,7’-difluorofluorescein; eosin-5-isothiocyanate; erythrosin 5-isothiocyanate; 6-(fluorescein-5-carboxamide) hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and -6)-carboxamide) hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon Green® 488 carboxylic acid, or succinimidyl ester; Oregon Green® 488 isothiocyanate; Oregon Green® 488-X succinimidyl ester; Oregon Green® 500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon Green® 514 carboxylic acid; Oregon Green® 514 carboxylic acid or succinimidyl ester; Rhodamine Green™ carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green™ carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green™-X succinimidyl ester or hydrochloride;RhodolGreen (trademark) carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl) sulfon rhodamine or di(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’-tetrabromosulfonfluorescein succinimidyl ester or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimidyl ester; 6-carboxytetramethylrhodamine succinimidyl ester; 5-(and-6)-carboxytetramethylrhodamine succinimidyl ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester; 6-carboxy-X-rhodamine succinimidyl ester; 5-(and-6)-carboxy-X-rhodamine succinimidyl ester; 5-carboxy-X-rhodamine triethylammonium salt; Lissamine (trademark) rhodamine B sulfonyl chloride; malachite green; isothiocyanate; NANOGOLD (registered trademark) mono(sulfosuccinimidyl ester); QSY (registered trademark) 21 carboxylic acid or succinimidyl ester; QSY (registered trademark) 7 carboxylic acid or succinimidyl ester; Rhodamine Red (trademark)-X succinimidyl ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamide) hexanoic acid; succinimidyl ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate;Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester; and X-rhodamine-5-(and -6) isothiocyanate are included.;

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

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

[0050] Selecting one or more suitable dyes based on the desired absorption or spectral excitation and emission characteristics of the particles is within the scope of ordinary skill in the art.

[0051] In some embodiments, multiple particles are used to determine the dynamic range and / or sensitivity of detection of a particular cell surface marker or combination thereof in a population of target cells. For example, a population of hydrogel particles can be adjusted to have the SSC and / or FSC profile of target cells, and a subpopulation of hydrogel particles is derivatized with a specific number of copies of a cell surface marker, such as a cell surface receptor, or a domain thereof, such as its epitope binding region. For example, individual subpopulations of hydrogel particles can each be derivatized to have a unique number of copies, e.g., one subpopulation contains 100 copies of a cell surface marker, a second subpopulation contains 1,000 copies of the same cell surface marker, a third subpopulation contains 10,000 copies of the same cell surface marker, and so on. The population of hydrogel particles is fluorescently stained for each cell surface marker, and the fluorescence is detected for the hydrogel particles of each subpopulation. In this regard, a standard curve of fluorescence emission for target cells having each cell marker can be generated using the subpopulations of hydrogel particles. The cell surface marker can be any of the cell surface markers provided by the present invention, or a binding region thereof, or a cell surface marker known to those of skill in the art.

[0052] "Substantially similar", as used herein, indicates at least 40% similarity, at least 50% similarity, at least 60% similarity, at least 70% similarity, at least 80% similarity, at least 90% similarity, at least 95% similarity, at least 96% similarity, at least 97% similarity, at least 98% similarity or at least 99% similarity.

[0053] In some embodiments, the refractive index (RI) of the disclosed hydrogel particles is 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 some embodiments, the refractive index (RI) of the disclosed hydrogel particles is from about 1.10 to about 3.0, or from about 1.15 to about 3.0, or from about 1.20 to about 3.0, or from about 1.25 to about 3.0, or from about 1.30 to about 3.0, or from about 1.35 to about 3.0, or from about 1.4 to about 3.0, or from about 1.45 to about 3.0, or from about 1.50 to about 3.0, or from about 1.6 to about 3.0, or from about 1.7 to about 3.0, or from about 1.8 to about 3.0, or from about 1.9 to about 3.0, or from about 2.0 to about 3.0.

[0055] In some embodiments, the refractive index (RI) of the disclosed hydrogel particles is 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 significantly measured compared to the SSC of target cells. In some embodiments, the disclosed hydrogel particles have an SSC 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 blood analyzer.

[0057] In some embodiments, the SSC of the hydrogel particles is modulated by incorporating a high refractive index molecule (or multiple high refractive index molecules) into the hydrogel. In some 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 some embodiments, the hydrogel particles of the present disclosure contain an alkyl acrylate and / or an alkyl methacrylate. The concentration of the monomer is adjusted in some embodiments to further adjust the refractive index of the hydrogel particles.

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

[0059] In some embodiments, the hydrogel particles of the present disclosure have a material modulus property (e.g., elasticity) that is more closely similar to the material modulus property of the target cells compared to polystyrene beads of the same diameter.

[0060] In some embodiments, a hemoglobin molecule or a dye molecule is covalently attached to a soluble hydrogel. Non-limiting examples of covalent linkers include disulfide linkers, ester linkers, amine linkers, thiol linkers, and carbonyl linkers. In some embodiments, a hemoglobin molecule or a dye molecule is non-covalently attached to a soluble hydrogel. Non-limiting examples of non-covalent linkers include streptavidin-biotin, neutravidin-biotin, and affinity tags such as His tag, GST tag, Halo tag, SNAP tag.

[0061] Shape / Size In some embodiments, the particles created (e.g., hydrogels) have the morphology or shape of the target cells. Mammalian red blood cells typically have the shape of a biconcave disk, with a flat and indented center, a dumbbell-shaped cross-section, and a toroidal rim at the edges of the disk. Red blood cells may have a shape characterized by a dimensionless "spherity index" of 4.84 × volume 2 / 3 / area. "Distribution of Size and Shape in Populations of Normal Human Red Cells," Canham and Burton, Circulation Research, Vol. XXII, March 1968, which is hereby incorporated by reference in its entirety for all purposes.

[0062] Artiodactyls (even-toed ungulates including cows, deer, and their relatives) have some exceptions with regard to shape, with small and highly elliptical red blood cells in llamas and camels (family Camelidae), very small spherical red blood cells in mouse deer (family Tragulidae), and red blood cells that take spindle-shaped, lanceolate, crescent-shaped, and other irregular polygonal and angular shapes in muntjacs and wapitis (family Cervidae), showing a wide variety of red blood cell morphologies.

[0063] In some embodiments, the particles created (e.g., hydrogels) are flexible and deformable to pass through very small capillaries.

[0064] The droplet size is related to the size of the microfluidic channel. The microfluidic channel can be of any size, for example, having a maximum dimension perpendicular to the 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 regulating the relative flow rates. In some embodiments, the droplet diameter is equivalent to the width of the channel or within about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the width of the channel.

[0066] The dimensions of the hydrogel particles of the present disclosure are substantially similar to the droplets in which they are formed. Thus, in some embodiments, the hydrogel particles have a diameter of less than about 1 μm, 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, or 1000 μm. In some embodiments, the hydrogel particles have a diameter greater than about 1 μm, 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, or 1000 μm.

[0067] In some embodiments, the dimensions (e.g., diameter, width, thickness) of the hydrogel particles of the present disclosure are substantially similar to the target cells being modeled. Thus, in some embodiments, the hydrogel particles have a diameter of less than about 1 μm, 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, or less than 1000 μm. In some embodiments, the hydrogel particles have a diameter greater than about 1 μm, 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, or greater than 1000 μm. In some embodiments, the hydrogel particles have a diameter in the range of 2.5 μm to 100 μm. In some embodiments, 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. Further, in some embodiments, the hydrogel particles have a width of less than about 1 μm, 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, or less than 1000 μm. In some embodiments, the hydrogel particles have a width greater than about 1 μm, 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, or greater than 1000 μm. In some embodiments, the hydrogel particles have a width in the range of 2.5 μm to 100 μm. In some embodiments, 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.Furthermore, in some embodiments, the hydrogel particles have a thickness of less than about 1 μm, less than 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, or 1000 μm. In some embodiments, the hydrogel particles have a thickness of greater than about 1 μm, greater than 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, or 1000 μm. In some embodiments, the hydrogel particles have a thickness in the range of 2.5 μm to 100 μm. In some embodiments, 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 some embodiments, each produced particle (e.g., hydrogel) is substantially similar in diameter or volume to a red blood cell, thereby resulting in produced particles having an impedance substantially similar to that of human red blood cells. Red blood cells typically have a diameter of 7-9 μm. 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 hereby incorporated by reference in its entirety for all purposes.

[0069] In some embodiments, the erythrocyte control composition comprises one or more populations of hydrogel particles, and at least one population of the hydrogel particles has an average diameter in the range of 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 some embodiments, at least one population of the hydrogel particles has an average diameter in the range of about 1 μm to about 15 μm. In some embodiments, at least one population of the hydrogel particles has an average diameter in the range of about 2 μm to about 15 μm. In some embodiments, at least one population of the hydrogel particles has an average diameter in the range of about 5 μm to about 15 μm. In some embodiments, at least one population of the hydrogel particles has an average diameter in the range of about 5 μm to about 10 μm. In some embodiments, at least one population of the hydrogel particles has an average diameter of about 7 μm to about 9 μm.

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

[0071] In some embodiments, the volume of the hydrogel particles of the present disclosure is substantially similar to the target cells being modeled. For example, if the target cells are red blood cells, the hydrogel particles can have a volume greater than about 1 fL, greater than 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, or 1000 fL. In some embodiments, the hydrogel particles have a volume in the range of 50 fL to 200 fL. In some embodiments, the hydrogel particles have a volume between about 75 fL and about 175 fL, between about 100 fL and about 150 fL, between about 110 fL and about 140 fL, between about 120 fL and about 130 fL, between about 122 fL and about 128 fL, between about 123 fL and about 127 fL, or between about 124 fL and about 126 fL. In some embodiments, the hydrogel particles have a volume between 75 fL and 175 fL. In some embodiments, the hydrogel particles have a volume between about 80 fL and about 100 fL. In some embodiments, the hydrogel particles have a volume between about 85 fL and about 95 fL. In some embodiments, the hydrogel particles have a volume between about 130 fL and about 170 fL. In some embodiments, the hydrogel particles have a volume between about 140 fL and about 160 fL. In some embodiments, the hydrogel particles have a volume between about 145 fL and about 155 fL.

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

[0073] In some embodiments, the hydrogel particles are formed by suspension polymerization, which is also referred to in the art as pearl polymerization, bead polymerization, or granular polymerization (see Elbert (2011). Acta Biomater. 7, pp. 31-56, which is hereby incorporated by reference in its entirety for all purposes). In suspension polymerization, the monomer is insoluble in the aqueous monomer solution in the continuous phase, e.g., the continuous oil phase. In suspension polymerization, polymerization initiation occurs within monomer-rich droplets, always with more than one radical per droplet. The monomer phase can include a difunctional monomer or monomers that can be monomer species (comonomers that can be difunctional monomers) in some embodiments. The monomer phase includes an initiator and / or a crosslinking agent in some embodiments.

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

[0075] In some embodiments, the hydrogel particles are formed by precipitation polymerization, as described, for example, in Elbert (2011). Acta Biomater. 7, pp. 31-56, which is hereby incorporated by reference in its entirety for all purposes. Precipitation polymerization is a technique that utilizes the difference in solubility between monomers and polymers to produce microparticles. Specifically, it is known that larger polymer chains generally have lower solubility than smaller polymer chains. Thus, phase separation can be advantageous when a certain molecular weight is exceeded. Precipitation polymerization begins first as solution polymerization in a single-phase homogeneous system. Immediately after the start of polymerization, in some embodiments, a relatively high concentration of polymer chains is present, promoting phase separation by nucleation. As the polymerization proceeds, the concentration of polymer chains decreases, and existing particles capture chains before new particle nucleation can occur. Thus, particle nucleation occurs only in a short period immediately after the start of the reaction, thereby resulting in a narrow size distribution of particles in some embodiments. Further methods include, but are not limited to, lithographic particle formation (Helgeson et al. (2011). Curr. Opin. Colloid. Interface Sci. 16, pp. 106-117, which is hereby incorporated by reference in its entirety for all purposes), membrane emulsification (e.g., by the micro-sieve emulsification technique described by Nanomi B.V. (Netherlands)), and microchannel emulsification (Sugiura et al. (2002). Languimir 18, pp. 5708-5712, which is hereby incorporated by reference in its entirety) and bulk emulsification (SNF Floerger available at snf.com.au / downloads / Emulsion_Handbook_E.pdf, which is hereby incorporated by reference in its entirety).

[0076] In some embodiments, the hydrogel particles are formed within a microfluidic device having two oil channels that converge into a central stream of an aqueous monomer solution. In this embodiment, droplets are formed at the interface between the two channels and the central stream, separating the droplets of the water-in-oil emulsion. Once the droplets are formed, in some embodiments, the droplets are stabilized prior to polymerization, for example, by adding a surfactant to the oil phase. However, in some embodiments, the droplets are not stabilized prior to polymerization. Polymerization of the monomer is, in some embodiments, induced by adding an accelerator (e.g., N,N,N’,N’-tetramethylethylenediamine) to one or both of the oil channels after the initial droplets are formed.

[0077] The aqueous monomer solution provided above can include a single monomer species or multiple monomer species. The aqueous monomer solution can include a comonomer, a difunctional monomer, or a combination thereof. In some embodiments, one monomer or multiple monomers can include a difunctional monomer, for example, one of the monomers described above. As described below, forward scattering or side scattering can be modulated by using a comonomer, for example, by adjusting the refractive index of the hydrogel particles.

[0078] In some embodiments, the central stream of the aqueous monomer solution includes a crosslinking agent, for example, N,N’-bisacrylamide. In further embodiments, the central stream of the aqueous monomer solution includes a crosslinking agent and an accelerator in addition to the monomer. In still further embodiments, the aqueous monomer solution includes an initiator, for example, an oxidizing agent, for example, ammonium persulfate.

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

[0080] The produced particles (e.g., hydrogels) can be fabricated and adjusted to regulate their electrical properties. Electrical properties can include, for example, capacitance, impedance, etc. The produced particles (e.g., hydrogels) can be fabricated to be useful in the calibration of a blood analyzer, for example, in the calibration of a Coulter counter. In some embodiments, the capacitance of the particles is adjusted by varying the amount of hydrogel monomer in the composition. For example, the concentrations of polyaniline, polyacetylene; polyphenylene vinylene; polypyrrole (X = NH) and polythiophene (X = S) comonomers; and polyaniline (X = NH / N) and polyphenylene sulfide (X = S) comonomers can all be adjusted to vary the capacitance. In some embodiments, the concentration of one or more of these monomers is increased to increase the capacitance of the hydrogel particles. In some embodiments, the diameter or volume of the produced particles (e.g., hydrogels) can be adjusted to mimic the impedance or capacitance of red blood cells, reticulocytes, or platelets.

[0081] Red blood cell control The erythrocyte control sample described in this specification can be a normal blood sample from a healthy human or adjusted to correspond to a medical condition. This can be achieved by (i) one or more populations of lysable hydrogel particles having an impedance substantially similar to that of human erythrocytes of average diameter, and (ii) adjusting the amount and / or ratio of a population of hemoglobin molecules or a population of dye molecules having an absorbance substantially similar to hemoglobin. For example, Table 1 below describes the following characteristics of a normal (healthy) blood sample measured by a blood 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 erythrocyte control composition of the present disclosure can match the characteristics of Table 1 and be adjusted to correspond to a normal (healthy) sample, or the control composition can be adjusted to correspond to various medical conditions by having characteristics outside the ranges shown in Table 1. For example, in some embodiments, the number, volume, and distribution of hydrogel particles in the control composition can be modified to correspond to the target RBC, MCV, and / or RDW, respectively. Similarly, in some embodiments, the concentration of hemoglobin or a dye having substantially similar absorbance can be modified to correspond to the target HGB and / or MCV. [Table 1]

[0082] (i) Normal erythrocyte characteristics In some embodiments, the erythrocyte control composition has an RBC (1×10 9 in the range of about 4.2 to about 5.9 1×10 9 (including all values and ranges therebetween)) of 9 / mL.

[0083] In some embodiments, the erythrocyte control composition has an HGB (g / dL) in the range of 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 therebetween). In some embodiments, the erythrocyte control composition for males has an HGB (g / dL) in the range of 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 some embodiments, the erythrocyte control composition for females has an HGB (g / dL) in the range of 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 some embodiments, the erythrocyte control composition has an MCV (fL) in the range of 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 therebetween).

[0085] In some embodiments, the erythrocyte control composition has an MCH (pg) in the range of about 28 to about 32 pg (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 therebetween).

[0086] In some embodiments, the erythrocyte control composition has an RDW (%) in the range of 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 therebetween).

[0087] In some embodiments, the erythrocyte control composition has NRBC that is lower than about 100 / 100 μL, for example, 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 sub-ranges therebetween). In some embodiments, the erythrocyte control composition has NRBC in the range of 99.5 to 20 / 100 μL, for example, 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 sub-ranges therebetween).

[0088] (ii) disease As discussed herein, control samples of the medical condition can be prepared by adjusting the content of the erythrocyte control.

[0089] In some embodiments, the composition has less than about 4.2 1×10 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 1×10 9 / mL (including all values and ranges therebetween) of RBC. In some embodiments, the composition has RBC in the range of about 1 to 4 1×10 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 1×10 9 / mL (including all values and ranges therebetween). In some embodiments, the composition has about 5.9 1×10 9more than, 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, or about 10 1×10 9 has RBCs greater than or equal to (including all values and ranges therebetween) 1×10 9 / mL. In some embodiments, the composition has from about 6 to about 10 1×10 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 1×10 9 / mL (including all values and ranges therebetween).

[0090] In some embodiments, the erythrocyte control composition (e.g., for females) has less than about 12 g / dL, for example, 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 of HGB (g / dL). In some embodiments, the erythrocyte control composition (e.g., for females) has from about 11.5 to about 6.5 g / dL, for example, 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 of HGB (g / dL).

[0091] In some embodiments, the erythrocyte control composition (e.g., for males) has an HGB (g / dL) of less than about 14 g / dL, such as 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 10.25, 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 or less than or equal to that (including all values and sub-ranges therebetween). In some embodiments, the erythrocyte control composition (e.g., for males) has an HGB (g / dL) in the range of about 13.5 to about 7.5 g / dL, such as 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 sub-ranges therebetween).

[0092] In some embodiments, the erythrocyte control composition (e.g., for males) has an HGB (g / dL) that exceeds about 17 g / dL, such as 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 therebetween). In some embodiments, the erythrocyte control composition (e.g., for males) has an HGB (g / dL) in the range of about 17.5 g / dL to about 22.5 g / dL, such as 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 therebetween).

[0093] In some embodiments, the erythrocyte control composition (e.g., for females) has an HGB (g / dL) that is greater than about 16 g / dL, such as 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 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 therebetween). In some embodiments, the erythrocyte control composition (e.g., for females) has an HGB (g / dL) in the range of about 16.5 g / dL to about 21.5 g / dL, such as 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 therebetween).

[0094] In some embodiments, the erythrocyte control composition has an MCV (fL) of less than 80 fL, such as 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 sub-ranges therebetween). In some embodiments, the erythrocyte control composition has an MCV (fL) in the range of about 79.5 to about 20 g / dL, such as 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 sub-ranges therebetween). In some embodiments, the erythrocyte control composition has an MCV (fL) greater than 100 fL, such as 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 sub-ranges therebetween). In some embodiments, the erythrocyte control composition has an MCV (fL) in the range of about 100.5 to about 200 g / dL, such as 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 sub-ranges therebetween).

[0095] In some embodiments, the erythrocyte control composition has a mean corpuscular hemoglobin (MCH) of less than 28 pg, such as 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 sub-ranges therebetween). In some embodiments, the erythrocyte control composition has an MCH (pg) in the range of about 27.5 to about 10 pg, such as 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 sub-ranges therebetween). In some embodiments, the erythrocyte control composition has an MCH of more than 32 pg, such as more 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 sub-ranges therebetween). In some embodiments, the erythrocyte control composition has an MCH (pg) in the range of about 32.5 to about 54 pg, such as 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 sub-ranges therebetween).

[0096] In some embodiments, the erythrocyte control composition has an RDW of less than 11%, such as, 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 sub-ranges therebetween). In some embodiments, the erythrocyte control composition has an RDW in the range of about 10.5% to about 5%, such as, 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 sub-ranges therebetween). In some embodiments, the erythrocyte control composition has an RDW greater than 16%, such as, for example, 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 sub-ranges therebetween). In some embodiments, the erythrocyte control composition has an RDW in the range of about 16.5% to about 22%, such as, for example, 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 sub-ranges therebetween).

[0097] In some embodiments, the erythrocyte control composition has an NRBC of greater than about 100 / 100 μL, such as, for example, 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 sub-ranges therebetween). In some embodiments, the erythrocyte control composition has an NRBC in the range of about 100.5 to about 150 / 100 μL, such as, 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 therebetween).

[0098] In some embodiments, the properties of the RBC control composition are modified to generate a positive control for the hematological detection of a condition or disease. In some embodiments, the condition or disease is elevated NRBC, myelodysplasia, anemia, RBC enzyme deficiency, red cell membrane disorder, hemoglobinopathy, polycythemia, hemochromatosis, hemoglobin increase, hemoglobin decrease, erythrocytosis, anisocytosis, macrocytosis, microcytosis, or leukemia. For example, in some embodiments, the average size of the hydrogel is reduced outside the detection range in healthy adults to generate a hematological reading that mimics the hematological readings of patients with microcytosis. In some embodiments, the average size of the hydrogel is increased outside the detection range in healthy adults to generate a hematological reading that mimics the hematological readings of patients with macrocytosis. In some embodiments, the size dispersity (RDW) of the hydrogel is increased outside the detection range in healthy adults to simulate the hematological readings of patients with nutritional deficiency or anisocytosis. In some embodiments, the concentration of the hydrogel is increased to simulate erythrocytosis. Exemplary RBC control compositions that mimic diseases are shown in FIGS. 8A and 8B.

[0099] In some embodiments, the concentration of hemoglobin (either human or of another animal, such as bovine) or a dye in the composition is reduced, with or without a change in the concentration of the hydrogel, to simulate the hematological readings of patients with iron deficiency anemia or hemoglobin decrease. In some embodiments, the concentration of hemoglobin (either human or of another animal, such as bovine) or a dye in the composition is increased, with or without a change in the concentration of the hydrogel, to simulate the hematological readings of patients with hemoglobin increase. Exemplary HGB mimics are shown in FIGS. 9A - 9B.

[0100] Whole blood sample control This specification also discloses a synthetic whole blood sample control. In some embodiments, the synthetic whole blood sample control includes (i) one or more populations of lytic hydrogel particles having an impedance substantially similar to that of human red blood cells of average diameter, (ii) a population of hemoglobin molecules or a population of dye molecules having an absorbance substantially similar to hemoglobin, and (iii) one or more populations of non-lytic hydrogel particles having an impedance substantially similar to that of human white blood cells of average diameter.

[0101] In some embodiments, one or more populations of non-lytic hydrogel particles are synthetic white blood cells. In some embodiments, the non-lytic hydrogel is composed of acrylamide and bisacrylamide. In some embodiments, one or more populations of non-lytic hydrogel particles have an average diameter in the range of about 12 μm to about 22 μm to mimic monocytes. In some embodiments, one or more populations of non-lytic hydrogel particles have an average diameter in the range of about 6 μm to about 18 μm to mimic lymphocytes. In some embodiments, one or more populations of non-lytic hydrogel particles have an average diameter in the range of about 8 μm to about 15 μm to mimic neutrophils. In some embodiments, one or more populations of non-lytic hydrogel particles have an average diameter in the range of about 12 μm to about 17 μm to mimic eosinophils. In some embodiments, one or more populations of non-lytic hydrogel particles have an average diameter in the range of about 10 μm to about 15 μm to mimic basophils.

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

[0103] Hydrogel As provided above, in embodiments of the present disclosure, the particles created include a plurality of polymers or copolymers. In some embodiments, the particles created include a plurality of hydrogel particles. A hydrogel is a material that contains a three-dimensional network of macromolecules that swells the hydrogel when water is present and shrinks when water is absent (or as the amount of water decreases) but does not dissolve in water. Swelling, i.e., water absorption, is the result of the presence of hydrophilic functional groups that are either bound to or dispersed within the macromolecular network. Crosslinking between adjacent macromolecules results in the water-insolubility of these hydrogels. Crosslinking can be due to chemical (i.e., by covalent bonds) or physical (i.e., van der Waals forces, hydrogen bonds, ionic force, etc.) bonds. These chemical crosslinks can also be hydrolyzed under certain conditions to reverse the insolubility of the hydrogel. Hydrogels prepared by synthesis can be prepared by polymerizing monomeric materials to form a backbone and crosslinking that backbone using a crosslinking agent. As referred to herein, the term "hydrogel" refers to a macromolecular material, whether dehydrated or in a hydrated state. A particularly valuable characteristic of a hydrogel is that the material retains its overall shape regardless of whether it is dehydrated or hydrated. Thus, a hydrogel that has an approximately spherical shape under dehydrating conditions is also spherical under hydrating conditions.

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

[0105] The particulate hydrogels provided herein are synthesized by polymerizing one or more of the monomers provided herein. The synthesis is carried out to form individual hydrogel particles. In some embodiments, a monomer material (monomer) is polymerized to form a homopolymer. However, in some embodiments, copolymers of different monomer units (i.e., comonomers) are synthesized and used in the methods provided herein. The monomers or comonomers used in the methods and compositions described herein are, in some embodiments, difunctional monomers or include difunctional monomers (when comonomers are used). In some embodiments, the hydrogel is synthesized in the presence of a crosslinking agent. In further embodiments, the hydrogel is synthesized in the presence of a polymerization initiator.

[0106] The amount of monomer can be varied, for example, to obtain properties that are substantially similar to certain optical or morphological properties of the target cells. In some embodiments, the monomeric component(s) (i.e., monomer, comonomer, bifunctional monomer, or combinations thereof, such as 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, succinimidyl 3-(2-pyridyldithio)propionate, dicumyl alcohol dimethacrylate, dicumyl alcohol diacrylate, 2,5-dimethyl-2,5-hexanediol dimethacrylate, acylhydrazone, or 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane (various crosslinking ratios), allylamine, or other comonomer or alginate that provides a chemical functional group for secondary labeling / conjugation) is present at about 10 weight percent to about 95 weight percent of the hydrogel. In further embodiments, the monomeric component(s) is present at about 15 weight percent to about 90 weight percent, or about 20 weight percent to about 90 weight percent of the hydrogel.

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

[0108] In some 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-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, derivatized versions thereof, or combinations thereof.

[0109] In some embodiments, 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, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, or combinations thereof.

[0110] In some embodiments, one or more of the following monomers are used herein to form an adjustable hydrogel: 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, 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-diphenylmethylacrylamide, N-(4-methylphenyl)methylacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylmethacrylamide, N-(4-methylphenyl)methylmethacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethacrylamide, N,N’-phenylphenyl ethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine (as described in U.S. Patent No. 6,657,030, which is hereby incorporated by reference in its entirety for all purposes).

[0111] Both synthetic monomers and biomonomers can be used in the hydrogels provided herein to form synthetic hydrogels, biogels, or hybrid hydrogels that contain synthetic and biological components (e.g., peptides, proteins, monosaccharides, disaccharides, polysaccharides, primary amines, sulfhydryls, carbonyls, carbohydrates, carboxylic acids present on biological molecules). For example, proteins, peptides, or carbohydrates can be used as individual monomers to form hydrogels with or without synthetic monomers (or polymers) in combination with chemically compatible comonomers and crosslinking chemistries (e.g., see Thermo Scientific Crosslinking Technical Handbook, titled "Easy molecular bonding crosslinking technology," available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf, the entire disclosure of which is hereby incorporated by reference in its entirety for all purposes). Compatible crosslinking chemistries include, but are not limited to, amines, carboxyls, and other reactive chemical side groups. Representative reactive groups suitable for use in the hydrogels and monomers described herein are provided in Table 2 below.

Table 2

[0112] In general, polymers can be formed using any form of polymerization chemistry / methods generally known to those skilled in the art. In some embodiments, the polymerization can be catalyzed by ultraviolet-induced radical formation and reaction progression. In other embodiments, the hydrogel particles of the present disclosure are produced by the polymerization of acrylamide or acrylate. For example, acrylamide is, in some embodiments, a polymerizable acrylamide derived from carbohydrates as described in U.S. Patent No. 6,107,365, the entire disclosure of which is incorporated herein by reference for all purposes. As described in that document and known to those skilled in the art, the specific binding of acrylamide groups to sugars readily adapts to various monosaccharides and higher-order polysaccharides, such as synthetic polysaccharides, or polysaccharides derived from natural sources such as glycoproteins found in serum or tissue.

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

[0114] In some embodiments, the hydrogel particles comprise a monofunctional monomer polymerized with at least one bifunctional monomer. By way of example, but not limited to, the formation of poly-acrylamide polymers using acrylamide and bis-acrylamide (a bifunctional monomer) can be mentioned. In some embodiments, the hydrogel particles presented herein comprise a bifunctional monomer polymerized with a second bifunctional monomer. By way of example, but not limited to, the formation of polymers using a mixed composition containing compatible chemistries, such as acrylamide, bis-acrylamide, and a wide range of additional chemistries, including bis-acrylamide structure analogs can be mentioned. The range of chemically compatible monomers, bifunctional monomers, and mixed compositions will be apparent to those skilled in the art and follow the principles of chemical reactions known to those skilled in the art. (See Thermo handbook and acrylamide polymerization handbook). For example, Thermo Scientific Crosslinking Technical Handbook titled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf), the entire disclosure of which is incorporated herein by reference for all purposes, and Polyacrylamide Emulsions Handbook (available at snf.com.au / downloads / Emulsion_Handbook_E.pdf) may be referred to.

[0115] In some embodiments, the hydrogel particles presented herein comprise a polymerizable monofunctional monomer, which is a monofunctional acrylic monomer. Non-limiting examples of monofunctional acrylic monomers for use herein include acrylamide; methacrylamide; N-alkylacrylamides, such as N-ethylacrylamide, N-isopropylacrylamide or N-tert-butylacrylamide; N-alkylmethacrylamides, such as N-ethylmethacrylamide or N-isopropylmethacrylamide; N,N-dialkylacrylamides, such as N,N-dimethylacrylamide and N,N-diethylacrylamide; N-[(dialkylamino)alkyl]acrylamides, such as N-[3-(dimethylamino)propyl]acrylamide or N-[3-(diethylamino)propyl]acrylamide; N-[(dialkylamino)alkyl]methacrylamides, 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 polymerize with the monofunctional monomers of the present disclosure to form the hydrogels described herein, which further contain a second functional group that can participate in a second reaction, such as the conjugation of a fluorophore or a cell surface receptor (or a domain thereof).

[0117] In some embodiments, the bifunctional 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 some embodiments, the hydrogel contains residues of the difunctional monomer, and the biofunctional monomer can dissolve the hydrogel in the presence of a lysis buffer. In some embodiments, the difunctional monomer contains bonds that can be cleaved by a lysis buffer. In some embodiments, the difunctional monomer contains disulfide bonds. In some embodiments, the difunctional monomer containing disulfide bonds 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 succinimidyl 3-(2-pyridyldithio)propionate. In some embodiments, the difunctional monomer contains acid-labile bonds. In some embodiments, the difunctional monomer is dicumyl alcohol dimethacrylate, dicumyl alcohol diacrylate, 2,5-dimethyl-2,5-hexanediol dimethacrylate, acylhydrazone, or 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane. In some embodiments, the acylhydrazone has the structure of

Chemical formula

Chemical formula

[0120] Higher order branched chains and linear comonomers can be substituted in the polymer mix to adjust the refractive index while maintaining the polymer density, as described in U.S. Patent No. 6,657,030, which is hereby incorporated by reference in its entirety for all purposes.

[0121] In some embodiments, the biomonomer is functionalized with acrylamide or acrylate. For example, in some embodiments, a polymerizable acrylamide-functionalized biomolecule is an acrylamide- or acrylate-functionalized protein (e.g., 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 the hydrogel monomer. In some embodiments, an acrylamide- or acrylate-functionalized monosaccharide, disaccharide, or polysaccharide is used as the polymerizable hydrogel monomer. In some embodiments, a structural polysaccharide is used as the polymerizable hydrogel monomer. In further embodiments, the structural polysaccharide is arabinoxylan, cellulose, chitin, or pectin. In some embodiments, alginic acid (alginate) is used as the polymerizable hydrogel monomer. In yet another embodiment, glycosaminoglycan (GAG) is used as the polymerizable monomer in the hydrogels provided herein. In further embodiments, the GAG is chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate, or hyaluronic acid (also known in the art as hyaluron or hyaluronate) that is used as the polymerizable hydrogel monomer. Further ranges of compatible biomonomers and their reactive chemistries are known to those of skill in the art and follow general principles of chemical reactivity.

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

[0124] Naturally occurring hydrogels useful in the present disclosure include various polysaccharides obtainable from natural sources such as plants, algae, fungi, yeast, marine invertebrates, and arthropods. Non-limiting examples include agarose, dextran, chitin, cellulose-based compounds, starch, derivatized starch, and the like. These generally have repeating glucose units as a major part of the polysaccharide backbone. Crosslinking chemistries for such polysaccharides are known in the art; see, for example, the Thermo Scientific Crosslinking Technical Handbook titled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf).

[0125] In some embodiments, hyaluronic acid is used as the hydrogel monomer (either as a single monomer or as a comonomer). Hyaluronic acid is functionalized with, for example, acrylate or acrylamide in some embodiments. Hyaluronic acid is a high molecular weight GAG composed of disaccharide repeating units of N-acetylglucosamine and glucuronic acid linked together via alternating β-1,4 and β-1,3 glycosidic bonds. In the human body, hyaluronate is found in several soft connective tissues including skin, umbilical cord, synovial fluid, and vitreous humor. Thus, in some embodiments where it is desired to mimic one or more optical properties of skin cells, umbilical cord cells, or vitreous humor cells, hyaluronic acid is used as the hydrogel monomer in some embodiments. Methods for fabricating hydrogel particles are described in Xu et al. (2012). Soft Matter. 8, pp. 3280-3294, the disclosure of which is incorporated herein by reference in its entirety for all purposes. As described in that literature, hyaluronic acid can be derivatized with various reactive handles depending on the desired cross-linking chemistry and other monomers used to form the 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 the hydrogel monomer (either as a single monomer or as a comonomer).

[0127] Other polysaccharides for use as hydrogel monomers or comonomers include, but are not limited to, agar, agarose, alginic acid, alguronic acid, alpha-glucan, amylopectin, amylose, arabinoxylan, beta-glucan, carrageenan, carrageenan polysaccharides (e.g., kappa, iota or lambda classes), cellodextrin, cellulins, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminoogalactan, gellan gum, glucan, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, icodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, pullulan, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, schizophyllan, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or combinations thereof. As described throughout, depending on the desired crosslinking chemistry and / or additional comonomers used in the hydrogel, the polysaccharide can be further functionalized. For example, one or more of the polysaccharides described herein are functionalized with acrylate or acrylamide in some embodiments.

[0128] In some embodiments, individual hydrogel particles or a plurality of hydrogel particles include a peptide, protein, protein domain, or combination thereof as one hydrogel monomer or a plurality of hydrogel monomers. In further embodiments, the protein is a structural protein or a domain thereof, such as silk, elastin, titin or collagen, or a domain thereof. In some embodiments, the protein is an extracellular matrix (ECM) component (e.g., collagen, elastin, proteoglycan). In still further embodiments, the structural protein is collagen. In still further embodiments, the collagen is type I collagen, type II collagen or type III collagen, or a combination thereof. In some embodiments, the hydrogel monomer includes a proteoglycan. In further embodiments, the proteoglycan is decorin, biglycan, testican, bikunin, fibromodulin, lumican, or a domain thereof.

[0129] In some embodiments, the acrylate-functionalized structural protein hydrogel monomer is used as a component of the hydrogels provided herein (e.g., an acrylate-functionalized protein or protein domain, such as silk, elastin, titin, collagen, proteoglycan, or a functionalized domain thereof). In further embodiments, the acrylate-functionalized structural protein hydrogel monomer includes a proteoglycan, such as decorin, biglycan, testican, bikunin, fibromodulin, lumican, or a domain thereof.

[0130] In some embodiments, as described in Lutolf et al. (2003). Proc. Natl. Acad. Sci. U.S.A. 100, 5413-5418, which is hereby incorporated by reference in its entirety for all purposes, PEG monomers and oligopeptides that can mimic extracellular matrix proteins are used in the hydrogels provided herein, for example, together with vinyl sulfone-functionalized multi-arm PEG, integrin-binding peptides, and bis-cysteine matrix metalloprotease peptides. In this particular embodiment, the hydrogel is formed by a Michael-type addition reaction between a dithiolated oligopeptide and a vinyl sulfone group on the PEG. The range of additional compatible chemical substances that can be incorporated here will be apparent to those skilled in the art and follows general principles of chemical reactivity. See, for example, the Thermo Scientific Crosslinking Technical Handbook titled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf).

[0131] Other bioactive domains in native 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 crosslinking domains can be used in the hydrogels provided herein. Details regarding the production of 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 hereby incorporated by reference in its entirety for all purposes.

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

[0133] A further range of biocompatible monomers that can be incorporated are known in the art; see, for example, the non-degradable biocompatible monomers disclosed by Shastri (2003). Current Pharmaceutical Biotechnology 4, pp. 331 - 337, which is hereby incorporated by reference in its entirety for all purposes. Other 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.

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

[0135] In some embodiments, the polymerization of the hydrogel is initiated by a persulfate or equivalent initiator that catalyzes radical formation. The range of compatible initiators is known to those skilled in the art and follows general chemical reaction principles. See, for example, the Thermo Scientific Crosslinking Technical Handbook titled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf). The 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 some embodiments, the persulfate is ammonium persulfate or potassium persulfate. In a further embodiment, the polymerization of the hydrogel provided herein is initiated by ammonium persulfate.

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

[0137] As discussed above, the hydrogel for use in the compositions and methods described herein can comprise any of the monomer units and crosslinking agents as described herein, and in one aspect is produced as hydrogel particles by polymerizing droplets (see, for example, FIG. 2). Microfluidic methods for producing a plurality of droplets including fluid droplets and hardened droplets are known to those skilled in the art and are described in U.S. Patent Application Publication No. 2011 / 0218123 and U.S. Patent No. 7,294,503, each of which is hereby incorporated 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, where the first fluid and the second fluid are substantially immiscible (e.g., droplets containing an aqueous-based liquid are substantially surrounded by an oil-based liquid).

[0138] Soluble Complete blood count (CBC) is a blood test used to assess health status and detect a wide range of disorders including anemia, infections, and leukemia. In a CBC test, several components including red blood cells, white blood cells, hemoglobin, hematocrit, and platelets are measured. Red blood cells in a sample are typically lysed to measure hemoglobin in the sample. Therefore, a control that is itself lysable is required.

[0139] A lysis buffer can use ammonium chloride. Blood lysis buffers often use ammonium chloride including 1× RBC lysis buffer and 10× RBC lysis buffer made by Thermo Fisher Scientific. Blood lysis buffers used for clinical blood samples are designed to lyse nucleated red blood cells and preserve white blood cells to perform white blood cell counting and quantitative measurement of hemoglobin. Other lysis buffers containing strong reducing agents such as dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), beta-mercaptoethanol (BME), or glutathione (GSH) can be designed to lyse the particles produced. Other lysis buffers can be designed to lyse acid-labile bonds. For example, the lysis buffer can contain formic acid. Other lysis buffers can be designed to lyse enzymatically degradable bonds. For example, the lysis buffer can contain chymotrypsin. As additional non-limiting examples, divalent ions such as ethylenediaminetetraacetic acid (EDTA) or citrate can be mentioned.

[0140] In some embodiments, the individual particle or particles comprise a biodegradable polymer as a monomer. In some embodiments, the biodegradable polymer is a poly(ester) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and copolymers thereof. These polymers can be degraded by hydrolysis and can dissolve macromolecular particles. In some embodiments, the biodegradable polymer is a carbohydrate or protein, or a combination thereof. For example, in some embodiments, monosaccharides, disaccharides or polysaccharides (e.g., glucose, sucrose, or maltodextrin), peptides, proteins (or domains thereof) are used as hydrogel monomers. Other biodegradable polymers include poly(hydroxyalkanoates) of the PHB-PHV class, additional poly(esters), as well as natural polymers such as modified poly(saccharides) such as starch, cellulose, and chitosan. In some embodiments, the biocompatible polymer is an adhesion protein, cellulose, carbohydrate, starch (e.g., maltodextrin, 2-hydroxyethyl starch, alginic acid), dextran, lignin, polyamino acid, amino acid, or chitin. Such biodegradable polymers are commercially available, for example, from Sigma Aldrich (St. Louis, MO).

[0141] In some embodiments, the protein comprises only natural amino acids. However, the present disclosure is not limited thereto. For example, self-assembling artificial proteins and proteins having non-natural amino acids (e.g., those incorporated into non-ribosomal peptides or synthetically introduced by synthetic methods, see, e.g., Zhang et al. (2013). Current Opinion in Structural Biology 23, pp. 581-587, which is incorporated herein by reference in its entirety for all purposes), or protein domains thereof, can also be used as hydrogel monomers. The range of non-natural (not natural) 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, which is incorporated herein by reference in its entirety for all purposes). The biodegradable polymer is, in some embodiments, used as a comonomer, i.e., in a mixture of monomers. The biodegradable polymer is, in some embodiments, a difunctional monomer.

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

[0143] In some embodiments, the degradation of individual hydrogel particles or a plurality of hydrogel particles can release the substances contained therein, whether by biodegradation means, dissolution, or other methods. For example, if the substance is a biomolecule, such as hemoglobin, degradation of the hydrogel particles containing hemoglobin can enable measurement of hemoglobin separately from the hydrogel monomers. In another example, if the substance is a biomolecule, such as hemoglobin, degradation of the hydrogel particles containing hemoglobin can be performed by a lysis buffer.

[0144] A plurality of fluid droplets (e.g., prepared using a microfluidic device) may be polydisperse (e.g., having various different sizes), or in some cases, the fluid droplets may be monodisperse or substantially monodisperse, e.g., having a homogeneous distribution of diameters, such that, as a result, about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% or less of the droplets have an average diameter 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 droplet population refers to the arithmetic mean of the diameters of the droplets. The average diameter of the particles can be measured, for example, by light scattering techniques. The average diameter of the hydrogel particles is, in some embodiments, adjusted, for example, by varying the flow rates of the first fluid and the second fluid streams in the channel(s) of the microfluidic device, or by varying the volume of the channel(s) of the microfluidic device.

[0145] Accordingly, 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 that includes at least one fluid channel with a cross-sectional dimension of less than 1 mm and a ratio of length to the maximum cross-sectional dimension perpendicular to the channel of at least about 3:1. Microfluidic devices that include microfluidic channels are particularly well-suited 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 WO2006 / 096571, U.S. Patent Application Publication No. 2007 / 0054119, U.S. Patent No. 7,776,927, and International Patent Application Publication WO2006 / 078841, each of which is incorporated herein by reference in its entirety for all purposes.

[0148] Functionalization (encapsulation, surface, and / or mobility) In some embodiments, beads, a plurality of beads, biomolecules, or a plurality of biomolecules are embedded (encapsulated) within hydrogel particles. Encapsulated beads or biomolecules are, in some embodiments, used to mimic one or more intracellular organelles of a target cell or a cell after ingestion of the particle. In some embodiments, encapsulation or embedding of the beads or biomolecules is achieved during the formation of the hydrogel particles. For example, the beads can be suspended at an appropriate concentration such that, on average, one bead is encapsulated / embedded within a single hydrogel particle. The bead suspension can be included, for example, within an aqueous solution of 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, for example, when hydrogel particles are formed by the method described above, in some embodiments, they can be further manipulated, for example, by embedding beads, a plurality of beads, biomolecules, or a plurality of biomolecules within the hydrogel particles.

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

[0151] In some embodiments, the encapsulated substance is an encapsulated molecule, such as a biomolecule. The biomolecule can be a single species or multiple different species. For example, proteins, peptides, carbohydrates, nucleic acids, or combinations thereof can be encapsulated within the hydrogel particles of the present disclosure. Additionally, different nucleic acid molecules (e.g., of various 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 since both forms of the biological material contain labile chemical side groups (or can be modified by commercial vendors (e.g., chemical side group modification by Integrated DNA Technology)). Such side groups are compatible with the reaction chemistries commonly found in comonomer compositions (e.g., acrylate chemistry, NHS-esters, primary amines, copper-catalyzed click chemistry (Sharpless)). The range of possible molecules that can be encapsulated and contain compatible chemistries is understood by those skilled in the art. In some embodiments, the encapsulated molecule may be one or more polynucleotides or analogs thereof. In some embodiments, the encapsulated molecule may be one or more polypeptides or analogs thereof. In some embodiments, the encapsulated molecule may be a complex of one or more polynucleotides and one or more polypeptides or analogs thereof. In some embodiments, the encapsulated molecule may be a combination of one or more polynucleotides, one or more polypeptides, and a complex of one or more polynucleotides and one or more polypeptides or analogs thereof.

[0152] In some embodiments, when the target cell is a red blood cell, the biomolecule encapsulated within the particle can be hemoglobin or a hemoglobin-like molecule, a dye, or an oxygen-carrying molecule, etc. Encapsulating hemoglobin within the hydrogel can include binding the hemoglobin to the hydrogel. When the hydrogel is amine-functionalized acrylamide, the hemoglobin can react with the modified amine group of the hydrogel via the sulfhydryl group (i.e., thiol group) of the cysteine of the hemoglobin. The hydrogel can be prepared for cross-linking with the thiol group of hemoglobin by treating it with a heterobifunctional cross-linking agent containing both succinimidyl ester and maleimide. Next, the maleimide-modified biomolecule can react with the thiol-containing hemoglobin to form a stable thioether cross-link.

[0153] In some embodiments, any one or more of hemoglobin or a hemoglobin-like molecule, a dye, or an oxygen-carrying molecule, an oxygen-transporting molecule, etc. may be present on the surface of the created particle. In some embodiments, the created particle contains one or more functional groups that can be used to bind any one or more of hemoglobin or a hemoglobin-like molecule, a dye, or an oxygen-carrying molecule, an oxygen-transporting molecule, etc. The functional group is, in some embodiments, an amine group, a carboxyl group, a hydroxy group, or a combination thereof. It should be understood that depending on the desired functionalization, multiple difunctional monomers can be used to functionalize the particle, for example, using different chemistries and different molecules.

[0154] In some embodiments, any one or more of hemoglobin or a hemoglobin-like molecule, a dye, or an oxygen-carrying molecule, an oxygen-transporting molecule, etc. may be present in solution together with the created particle. In this embodiment, the created population of cells can mimic a CBC test in which red blood cells are lysed and hemoglobin is not present within the red blood cells and can serve as a control therefor.

[0155] subpopulation In some embodiments, different sub-populations of the produced particles are made, each having a different concentration of a biomolecule. In further embodiments, the biomolecule is any one or more of hemoglobin or hemoglobin-like molecules, dyes, or oxygen-carrying molecules, oxygen-transporting molecules, etc. In further embodiments, the biomolecule is a nucleic acid, a protein, an intracellular ion, such as calcium acid (or other biomolecule of the user's choice, such as calcium). In some embodiments, different sub-populations of hydrogel particles are made, each having a different concentration of a drug substance. The drug substance is, in some embodiments, 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., a type I / II / III polyketide, a non-ribosomal peptide having bioactive properties, or other small molecule entity commonly classified by those skilled in the art).

[0156] In this regard, the present disclosure is particularly useful for determining the assay resolution at which cells are stained for their respective nucleic acid or protein content. In some embodiments, different populations of the produced particles provided herein are encapsulated with one or more of known different amounts of hemoglobin or hemoglobin-like molecules, dyes, or oxygen-carrying molecules, oxygen-transporting molecules, etc. The individual produced particles are stained for intracellular substances, and fluorescence is measured by a blood analyzer for the individual particles of the various populations. Thereby, a standard curve can be created to establish the sensitivity and dynamic range of the intracellular assay. Once established, a sample can be passed through a cytometer to detect target cells, if present, and quantify the amount of intracellular substance in each target cell. In some embodiments, the encapsulated substance is hemoglobin.

[0157] In some embodiments, the methods provided herein are used to determine the sensitivity and / or dynamic range of a blood quantification assay. In this embodiment, the sample is examined for the cell type (if present) within the sample and the amount of biomolecule within the cell.

[0158] In some embodiments, the present disclosure provides means for determining the resolution and / or sensitivity of a hematological assay. Hydrogel particles, in some embodiments, encapsulate one or more of a known amount of hemoglobin or hemoglobin-like molecules, dyes, or oxygen-carrying molecules, oxygen transport molecules, etc. at various concentrations and can then be stained with an appropriate dye. Fluorescence is measured for the various particles to determine the sensitivity and / or dynamic range of the assay. The fluorescence values are then compared to values obtained from cells in the sample to determine whether target cells are present, whether the target cells contain intracellular proteins, and the amount of protein.

[0159] In some embodiments, individual hydrogel particles are engineered to have at least one optical and / or morphological property similar to that of red blood cells. The individual particles are embedded with a known amount of the biomolecule of interest, such as hemoglobin. The particles are used to create a standard curve for a biomolecule detection assay for a specific cell type.

[0160] Method In some embodiments, a method is provided for calibrating a blood analyzer for the analysis of target cells. In some embodiments, the method includes (a) inserting into the device an erythrocyte control composition having properties substantially similar to those of the target cells, (b) using the blood analyzer to measure the properties of the erythrocyte control composition, (c) comparing the measured properties of the erythrocyte control composition to the target sample, and (b) optionally adjusting the blood analyzer so that the properties of the erythrocyte control composition are substantially similar to the properties of the target sample, thereby calibrating the blood analyzer for the analysis of human whole blood samples.

[0161] In some embodiments, a method is provided for evaluating a red blood cell sample using a blood analyzer. In some embodiments, the method includes: (a) measuring one or more characteristics of a red blood cell control composition; (b) measuring one or more characteristics of a red blood cell sample; and (c) comparing the measured values of the red blood cell sample with the measured values of the control sample.

[0162] In some embodiments, one or more populations of dissolvable hydrogel particles have the characteristics of normal red blood cells, as disclosed in the "Red Blood Cell Control" section of this application. In some embodiments, a deviation of the measured values of the red blood cell sample from the measured values of the red blood cell control composition indicates a condition or disease.

[0163] In some embodiments, one or more populations of dissolvable hydrogel particles have the characteristics of diseased red blood cells, as disclosed in the "Red Blood Cell Control" section of this application. In some embodiments, a substantial similarity between the measured values of the red blood cell sample and the measured values of the control sample indicates a condition or disease.

[0164] Blood analyzers are known in the art and include commercially available devices for performing flow cytometry. Blood analyzers can use, for example, aperture-based techniques, image-based techniques, and / or waveform-based techniques, among others. In some embodiments, the blood analyzer is an optical-based cytometer. In some embodiments, the blood analyzer is an electrical impedance-based cytometer.

Examples

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

[0166] (Example 1) Generation of Hydrogel Particles The photomask for UV lithography was obtained from CADart Services Inc. and designed using AutoCad (AutoDesk, Inc.). The SU-8 photoresist (Microchem, Inc.) was photocrosslinked on a 4-inch silicon wafer using a collimated UV light source (OAI, Inc.) to generate a master for fabricating microfluidic devices. PDMS (polydimethylsiloxane, Sigma Aldrich, Inc.) was prepared and formed using standard methods published for soft lithography and the fabrication of microfluidic devices (see McDonald JC, et al., 2000, Electrophoresis 21 :27-40).

[0167] The droplets were formed using a flow focusing geometry where two oil channels converge into a central stream of an aqueous monomer solution to separate the droplets of the water-in-oil emulsion. Fluorocarbon oil (Novec 7500 3M, Inc.) was used as the outer continuous phase liquid for droplet formation. To stabilize the droplets before polymerization, a surfactant was added to the oil phase at 0.5 w / w% (Krytox 157 FSH, an ammonium carboxylate salt from Dupont). To produce basic polyacrylamide gel particles, a central phase of an aqueous monomer solution containing N-acrylamide (1 - 20 w / v%), a crosslinking agent to dissolve the hydrogel (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 succinimidyl 3-(2-pyridyldithio)propionate, dicumyl alcohol dimethacrylate, dicumyl 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%) were used. To induce polymerization of the hydrogel particles after droplet formation, an accelerator (N,N,N’,N’-tetramethylethylenediamine (2% vol%)) was added to the oil phase.

[0168] Comonomers can be added to the basic gel formulation to add functionality. Allyl-amine provided primary amine groups for secondary labeling after gel formation. Forward scattering can be modulated by adjusting the refractive index of the gel by adding allyl acrylate and allyl methacrylate comonomers. The side scattering of the 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] The stoichiometric multiplexing of hydrogel particles was achieved by utilizing comonomers containing chemically orthogonal side groups (amines, carboxyls, maleimides, epoxides, alkynes, etc.) for secondary labeling.

[0170] Droplets were formed at an average rate of 5 kHz and collected in a fluorocarbon oil phase. Polymerization was completed at 50 °C in 30 minutes, and the resulting hydrogel particles were washed from the oil into an aqueous solution.

[0171] (Example 2) Generation and visualization of insoluble hydrogel particles Water containing 5% acrylamide, 0.25% bisacrylamide, 0.05% allylamine, and 0.1% ammonium persulfate was flowed into the central channel as shown in Figure 3A, and oil containing 0.1% TEMED was focused through a 10-μm nozzle to produce 10-μm hydrogel particles. After polymerization, the particles were washed with water and conjugated with the desired dye as shown in Figure 3B. The fluorescent hydrogel particles were visualized with a fluorescence microscope as shown in Figure 3C. The size of the hydrogel can be adjusted by increasing or decreasing the size of the nozzle.

[0172] (Example 3) Generation of RBC mimics Hydrogel particles can be adjusted to various sizes and / or polymer contents. As shown in Figures 7B - 7D, exemplary RBC mimics had a Coulter volume corresponding to a diameter in the range between 3 μm and 8 μm, a Coulter volume corresponding to a diameter of approximately 12 μm but with a low polymer weight percentage (reducing the distribution), or a Coulter volume corresponding to a diameter of approximately 12 μm but with a high polymer weight percentage (increasing the distribution). The Coulter volume measured by the Coulter principle can be regarded as the perceived volume. However, the actual diameter of each of these RBC mimics may vary according to the porosity of the fabricated particles as indicated by the polymer weight percentage.

[0173] Different disease patterns can be mimicked by different sizes and / or polymer contents of hydrogel particles. Figures 8A - 8B show further examples where the RBC mimics were adjusted to mimic RBCs in microcythemia, macrocythemia, or anisocytosis.

[0174] (Example 4) Generation of HGB mimics Human HGB mimics can be generated using a red dye (Allura Red or any dye having a spectrum similar to hemoglobin). Aqueous solutions of Allura Red were prepared at different concentrations and analyzed with a Beckman Coulter DxH 690T hematology analyzer. As shown in Figure 9A, there was a linear relationship between the concentration of Allura Red and the concentration of human HGB, and any specific hemoglobin-related diseases could be accurately reproduced. At 5 mg / mL of Allura Red, the results from the analyzer were similar to those of normal human whole blood. At low concentrations of Allura Red, the results were similar to those of patients with hypochromia or anemia. At high concentrations of Allura Red, the results were similar to those of patients with hyperchromia.

[0175] HGB derived from animals other than humans can also be used to generate human HGB mimics. Bovine HGB solutions at different concentrations were prepared and analyzed with a Beckman Coulter DxH 690T hematology analyzer. As shown in Figure 9B, there was a linear relationship between the absorbance of bovine HGB and that of human HGB, and any specific hemoglobin-related diseases could be accurately reproduced as discussed in the above paragraph.

[0176] (Example 5) Hemolysis of hydrogel particles A chemically or physically labile crosslinker or cleavable polymer chain can be incorporated into the hydrogel polymer matrix to mimic hemolysis of red blood cells induced by salts or surfactants.

[0177] In this example, hydrogel particles were synthesized by copolymerizing acrylamide with bis(2-methacryloyl)oxyethyl disulfide, which acts as a crosslinking agent that can be cleaved by redox. The scattering properties and Coulter volume of the hydrogel particles were adjusted to match those of human-derived red blood cells. The disulfide was reduced to thiol and the crosslinking agent was cleaved when exposed to a 5 wt% solution of 1,4-dithiothreitol (DTT) or beta-mercaptoethanol (BME) (e.g., 2-mercaptoethanol).

[0178] As a result, the hydrogel matrix was decomposed into water-soluble single polyacrylamide chains and dissolved. After dissolution, the hydrogel particles could no longer be detected by flow cytometry or by the Coulter principle. Therefore, the dissolved particles did not interfere with the measurement of other more stable particles, such as white blood cells or their mimics, that may be present in the same suspension.

[0179] (Example 6) Compatibility test of hydrogel lysis buffer with whole blood measurement This example aims to test whether the hydrogel lysis buffer can affect the measurement of whole blood cell samples by a blood 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 blood analyzer to measure the characteristics of the whole blood sample. As shown in Figure 10, DTT did not affect the measurement. Approximately 2 minutes after adding DTT, the red blood cell control composition diffused and dissolved in a static state.

[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 descriptions, and protocols cited throughout this application are hereby incorporated by reference in their entirety for all purposes.

[0182] The embodiments illustrated and discussed herein are intended only to teach those skilled in the art the best mode known to the inventors for making and using the invention of the present disclosure. As will be understood by those skilled in the art in light of the foregoing teachings, modifications and variations of the foregoing embodiments of the present disclosure are possible without departing from the spirit of the invention. Therefore, it is understood that within the scope of the claims and their equivalents, the invention of the present disclosure may be practiced in a manner different from that particularly described.

Claims

1. (i) A collection of soluble hydrogel particles having an impedance substantially similar to that of a human red blood cell of average diameter, (ii) a group of hemoglobin molecules and / or a group of dye molecules having an absorbance substantially similar to that of hemoglobin, (iii) A group of insoluble hydrogel particles having an impedance substantially similar to that of a human leukocyte of average diameter, Synthetic whole blood sample control, including

2. At least one hemoglobin molecule or pigment molecule a. Encapsulated within a group of soluble hydrogel particles, b. Bound to a group of soluble hydrogel particles, c. Covalently bonded to a group of soluble hydrogel particles, or d. Non-covalently bonded to a group of soluble hydrogel particles, The synthetic whole blood sample control according to claim 1.

3. The synthetic whole blood sample control according to claim 1, wherein the synthetic whole blood sample control is a solution, in which a group of hemoglobin molecules or pigment molecules are suspended or dissolved, and a group of soluble hydrogel particles and / or insoluble hydrogel particles are suspended in the solution.

4. The synthetic whole blood sample control according to claim 1, wherein the population of soluble hydrogels comprises polymerized monomers and comonomers, the comonomer being a difunctional monomer, and the difunctional monomer comprising a bond that can be dissolved by a lysis buffer.

5. The synthetic whole blood sample control according to claim 4, wherein the bifunctional monomer comprises a disulfide bond, an acid-unstable bond, an enzymatically degradable bond, or a combination thereof.

6. The synthetic whole blood sample control according to claim 5, wherein the difunctional monomer comprises a disulfide bond, and the difunctional monomer is selected from the group consisting of 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, succinimidyl 3-(2-pyridyldithio)propionate, and combinations thereof.

7. The synthetic whole blood sample control according to claim 5, wherein the disulfide bond can be dissolved by a lysis buffer selected from the group consisting of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), beta-mercaptoethanol (BME), glutathione (GSH), and combinations thereof.

8. The synthetic whole blood sample control according to claim 5, wherein the difunctional monomer contains an acid-unstable bond, and the difunctional monomer is selected from the group consisting of dicumyl alcohol dimethacrylate, dicumyl alcohol diacrylate, 2,5-dimethyl-2,5-hexanediol dimethacrylate, acylhydrazone, 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane, and combinations thereof.

9. The synthetic whole blood sample control according to claim 5, wherein the acid-unstable binding can be dissolved by a lysis buffer containing formic acid.

10. The synthetic whole blood sample control according to claim 4, wherein the lysis buffer comprises ethylenediaminetetraacetic acid (EDTA), citrate, or potassium persulfate.

11. Monomers include lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, 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, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acylate, phenyl methacrylate, benzyl acylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate Rate, 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, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl N-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-dibenzyl methacrylamide, N-diphenylmethylacrylamide, N-(4-methylphenyl)methylacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylmethacrylamide, N-(4-methylphenyl)methylmethacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenyl A synthetic whole blood sample control according to claim 4, selected from the group consisting of nylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethacrylamide, N,N'-phenylphenylethylmethacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridinemethacrylamide, N-alkylacrylamide, N-alkylmethacrylamide, N,N-dialkylacrylamide, N-[(dialkylamino)alkyl]acrylamide, N-[(dialkylamino)alkyl]methacrylamide, (dialkylamino)alkyl acrylate, (dialkylamino)alkyl methacrylate, and combinations thereof.

12. The synthetic whole blood sample control according to claim 4, wherein the comonomer is selected from the group consisting of allylamine, allyl acrylate, allyl methacrylate, allyl alcohol, allyl isothiocyanate, allyl chloride, allyl maleimide, bis-acrylamide, and combinations thereof.

13. The synthetic whole blood sample control according to claim 12, wherein the bis-acrylamide is selected from the group consisting of N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, N,N'-ethylenebis-methacrylamide, N,N'-propylenebisacrylamide, and N,N'-(1,2-dihydroxyethylene)bisacrylamide.

14. The synthetic whole blood sample control according to claim 1, further comprising one or more additional molecules encapsulated within at least one soluble hydrogel particle, wherein the one or more additional molecules are selected from the group consisting of polynucleotides, polypeptides, and combinations thereof.

15. (i) A group of soluble hydrogel particles having an average diameter in the range of approximately 1 μm to approximately 20 μm, (ii) The synthetic whole blood sample control has a higher HBG than 17 g of hemoglobin per dL, as measured by absorbance, and / or (iii) A population of hemoglobin molecules is contained within a population of soluble hydrogel particles, and the population of soluble hydrogel particles contains mean corpuscular hemoglobin (MCH) in the range of approximately 28 pg to approximately 32 pg when measured using impedance-based cytometry. The synthetic whole blood sample control according to claim 1.

16. The synthetic whole blood sample control according to claim 1, wherein a group of dye molecules having an absorbance substantially similar to that of hemoglobin has a peak excitation wavelength in the range of about 600 nm to about 750 nm and / or a peak emission wavelength in the range of about 625 nm to about 775 nm.

17. A method for calibrating a blood analyzer for the analysis of a target whole blood sample containing red blood cells and white blood cells, (a) Inserting a synthetic whole blood sample control into a blood analyzer, wherein the synthetic whole blood sample control is (i) A collection of soluble hydrogel particles having an impedance substantially similar to that of a human red blood cell of average diameter, (ii) a group of hemoglobin molecules and / or a group of dye molecules having an absorbance substantially similar to that of hemoglobin, (iii) A group of insoluble hydrogel particles having an impedance substantially similar to that of a human leukocyte of average diameter, The steps include, (b) Using a blood analyzer, measure the impedance of a population of soluble hydrogel particles and a population of insoluble hydrogel particles, and (c) Calibrate the blood analyzer for red blood cell analysis based on the impedance of the measured population of soluble hydrogel particles. A step of calibrating the blood analyzer for leukocyte analysis based on the impedance of the measured population of insoluble hydrogel particles, The above method, including.

18. (d) A step of bringing a synthetic whole blood sample control into contact with a lysis buffer, wherein the group of soluble hydrogel particles comprises a bifunctional monomer having a bond that can be dissolved by the lysis buffer, (e) A step of measuring the impedance of a population of insoluble hydrogel particles using a blood analyzer, and (f) A step of calibrating the blood analyzer for the analysis of leukocytes in a target whole blood sample based on the measured impedance. The method according to claim 17, including the method described in claim 17.

19. The method according to claim 17, comprising the step of measuring the concentration of hemoglobin molecules and / or pigment molecules using a blood analyzer, and adjusting the concentration of hemoglobin molecules and / or pigment molecules based on the concentration of hemoglobin molecules in a target whole blood sample.

20. A method for evaluating a target whole blood sample containing red blood cells and white blood cells, (a) Inserting a synthetic whole blood sample control into a blood analyzer, wherein the synthetic whole blood sample control is (i) A collection of soluble hydrogel particles having an impedance substantially similar to that of a human red blood cell of average diameter, (ii) a group of hemoglobin molecules and / or a group of dye molecules having an absorbance substantially similar to that of hemoglobin, (iii) A group of insoluble hydrogel particles having an impedance substantially similar to that of a human leukocyte of average diameter, The steps include, (b) A step of measuring the impedance of a population of soluble hydrogel particles and comparing the impedance of red blood cells in a target whole blood sample with the impedance of a population of soluble hydrogel particles in a synthetic whole blood sample control. (c) The step of bringing a synthetic whole blood sample control into contact with a lysis buffer, wherein a collection of soluble hydrogel particles comprises a bifunctional monomer having a bond that can be dissolved by the lysis buffer, and (d) A step of measuring the impedance of a population of insoluble hydrogel particles and comparing the impedance of leukocytes in a target whole blood sample with the impedance of a population of insoluble hydrogel particles in a synthetic whole blood sample control. The above method, including.

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