Analysis cartridges and methods of using them

Abstract

Analysis cartridges for analyzing cellular and non-cellular constituents of a biological sample are provided. A cellular analysis module can comprise an imaging chamber comprising a top panel and a bottom panel separated by a suitable distance to produce a monolayer of cells between the panels. A chemical analysis module can comprise a sample distribution channel that distributes a non-cellular portion of a biological sample into a plurality of detection chambers that analyze a plurality of non-cellular constituents. The cellular analysis module can be stacked above the chemical analysis module and vice versa or the two modules can be in substantially the same horizontal plane. A filtration module filters the biological sample as the biological sample transfers from the cellular analysis module through the filter to the chemical analysis module. Methods of analyzing a biological sample using the cartridges disclosed herein are also provided.

Description

ANALYSIS CARTRIDGES AND METHODS OF USING THEMCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit to the filing date of U.S. Provisional Patent Application Serial No. 63 / 692,593, filed on September 9, 2024, the disclosure of which application is herein incorporated by reference in its entirety.INTRODUCTION

[0002] Analyses of biological samples, such as blood samples, are limited by the availability of samples. Also, analyses of biological samples, such as blood samples often involve testing cells as well as non-cellular constituents. Therefore, analyses of biological samples, particularly for their cells and non-cellular constituents, using the smallest possible amounts are desirable.SUMMARY

[0003] In certain aspects, the disclosure provides analysis cartridges that allow analyzing small volumes of biological samples. In certain embodiments, the analysis cartridges allow analyses of cells as well as non-cellular portions of biological samples.

[0004] In certain aspects, the disclosure provides an analysis cartridge for analyzing a biological sample, the analysis cartridge comprising:

[0005] a cellular analysis module for analyzing cells of the biological sample,

[0006] a chemical analysis module for analyzing non-cellular constituents of the biological sample, and

[0007] a filtration module that filters the biological sample by transferring the biological sample from the cellular analysis module through a filter to the chemical analysis module thereby delivering the non-cellular portion of the biological sample to the chemical analysis module.

[0008] In some cases, the disclosure provides an analysis cartridge, comprising:

[0009] a cellular analysis module and a chemical analysis module, wherein the cellular analysis module is stacked above the chemical analysis module and the cellular analysis module and the chemical analysis module are connected by a filtration module. In certain such cases, the filtration module filters the biological sample as the biological sample transfers, under the force of gravity, from the cellular analysis module through the filter to the chemical analysis module.

[0010] In certain other cases, the chemical analysis module is stacked above the cellular analysis module and the filtration module filters the biological sample as the biological sample transfers, under a force that moves the biological sample against the force of gravity, from the cellular analysis module through the filter to the chemical analysis module. A biological sample can bepushed upwards through the filter, for example, via a force generator, such as a pump, as it is filtered to produce non-cellular constituents of the biological sample.

[0011] A biological sample delivered to a cellular analysis module is analyzed in the cellular analysis module for cells of the biological sample. In certain embodiments, the biological sample is blood and the cellular analysis module analyzes the blood for complete blood count (CBC).

[0012] A non-cellular portion of a biological sample, such as plasma, is filtered and delivered to the chemical analysis module and is analyzed in the chemical analysis module for non-cellular constituents of the biological sample. In certain embodiments, the biological sample is blood and the chemical analysis module analyzes plasma for comprehensive metabolic panel (CMP).

[0013] In certain aspects, the disclosure provides an analysis cartridge, wherein the cellular analysis module and the chemical analysis module are in substantially the same horizontal plane and are connected by a filtration module. In certain such cases, the filtration module filters the biological sample as the biological sample transfers under a force that moves the biological sample from the cellular analysis module through the filter to the chemical analysis module thereby delivering a non-cellular portion of the biological sample to the chemical analysis module.

[0014] The filtration module can have a membrane or filter for filtering cells from the non-cellular portion of the biological sample. For example, blood can be pushed through the filter, for example, under the force of gravity, a pump or a vacuum, as it is filtered to produce plasma.

[0015] In some embodiments, the cellular analysis module comprises an imaging chamber comprising a top panel and a bottom panel separated by a suitable gap to produce a monolayer of cells between the top and the bottom panels. In some cases, the imaging chamber comprises:

[0016] a sample delivery edge where the gap between the top panel and the bottom panel is accessible to the biological sample delivered to the sample delivery edge, and

[0017] a sample loading port configured to deliver the biological sample to the sample delivery edge thereby allowing the biological sample to fill the imaging chamber.

[0018] In certain cases, a chemical analysis module comprises a sample distribution channel that distributes a non-cellular portion of the biological sample into a plurality of detection chambers that detect a plurality of non-cellular constituents. The detection chambers can contain reagents that analyze specific constituents within the non-cellular portion of the biological sample.

[0019] In certain embodiments, a chemical analysis module comprises a top panel and a bottom panel and a plurality of channels disposed between the top panel and the bottom panel, wherein the plurality of channels are configured for analyzing a plurality of non-cellular constituents. The plurality of channels comprise reagents that analyze specific constituents within the non-cellular portion of the biological sample.

[0020] Also provided herein are methods of analyzing in the analysis cartridges provided herein a biological sample, such as blood, for cells as well as non-cellular constituents. Further provided herein are analyzers that use the analysis cartridges provided herein to analyzer a biological sample, such as blood, for cells and non-cellular constituents.BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1 depicts an exploded view of an exemplary cellular analysis module. The cellular analysis module comprises an imaging chamber. The cellular analysis module also comprises a sample chamber and a sample loading port. The sample loading port delivers the biological sample to the sample delivery edge of the imaging chamber thereby allowing the biological sample to load into the imaging chamber.

[0022] FIG. 2 depicts an exploded view of an exemplary chemical analysis module. This module comprises a filtration module, a sample metering chamber, diluent metering chamber, and diluent mixing chamber. The chemical analysis module also comprises a sample distribution channel that distributes a non-cellular portion of a biological sample into a plurality of detection chambers. The plurality of detection chambers detect and / or quantify a plurality of non-cellular constituents.

[0023] FIG. 3 depicts a top view of an exemplary analysis cartridge. This cartridge comprises a cellular analysis module stacked above a chemical analysis module with a filtration module that filters a biological sample.

[0024] FIG. 4 depicts a side view of an exemplary analysis cartridge. This cartridge comprises a cellular analysis module stacked above a chemical analysis module with a filtration module that filters a biological sample.

[0025] FIG. 5 depicts an exemplary analysis cartridge where a cellular analysis module and a chemical analysis module are located in substantially the same horizontal plane with a filtration module that filters a biological sample.DETAILED DESCRIPTION

[0026] Certain aspects of the present disclosure provide analysis cartridges that allow analyzing cells as well as non-cellular constituents of a biological sample, such as blood. In certain embodiments, an analysis cartridge comprises a cellular analysis module for analyzing cells of a biological sample and a chemical analysis module for analyzing non-cellular constituents of the biological sample.

[0027] Also provided herein are methods of analyzing a biological sample, such as a blood sample in the analysis cartridges provided herein. Further provided herein are analyzers that use theanalysis cartridges provided herein to analyzer a biological sample, such as blood, for cells and non-cellular constituents.

[0028] Before the present analysis cartridges, methods, and analyzers are described in greater detail, it is to be understood that the present disclosure is not limited to particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0029] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the analysis cartridges and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the analysis cartridges and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the analysis cartridges and methods.

[0030] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating un-recited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0032] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein. In the following specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings.

[0033] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and“the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth.

[0034] The term “comprising” is used herein as requiring the presence of the named component and allowing the presence of other components. The term “comprising” should be construed to include the term “consisting essentially of’ and “consisting of.” The “consisting essentially of” allows the presence of the named component(s), along with other components which do not change the function / structure of the named component(s). The “consisting of’ allows the presence of the named component(s), along with any adhesives or other bonding means for attaching the listed component(s).

[0035] Numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0036] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0037] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 2 grams to 10 grams” is inclusive of the endpoints, 2 grams and 10 grams, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0038] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. When used in the context of a range, the modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range of from about “2 to about 10” also discloses the range “from 2 to 10.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9- 1.1.

[0039] It should be noted that many of the terms used herein are relative terms. For example, the terms “upper” and “lower” are relative to each other in location, i.e., an upper component is located at a higher elevation than a lower component in a given orientation, but these terms can change if the component is flipped. The terms “inlet” and “outlet” are relative to a fluid flowing throughthem with respect to a given structure, e.g., a fluid flows through the inlet into the structure and flows through the outlet out of the structure.

[0040] The terms “horizontal” and “vertical” are used to indicate direction relative to an absolute reference, i.e., ground level. However, these terms should not be construed to require structures to be absolutely parallel or absolutely perpendicular to each other. For example, the first vertical structure and the second vertical structure are not necessarily parallel to each other. The terms “top” and “bottom” are used to refer to surfaces where the top is always higher than the bottom relative to an absolute reference, i.e., the surface of the earth. The terms “upwards” and “downwards” are also relative to an absolute reference; upwards is always against the gravity of the earth while downwards is always towards the gravity of the earth.

[0041] The term “parallel” should be construed in its lay sense of two surfaces that maintain a generally constant distance between them, and not in the strict mathematical sense that such surfaces will never intersect when extended to infinity.

[0042] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0043] The claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only,” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0044] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present analysis cartridges and methods. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.AN LYSIS CARTRIDGES

[0045] As summarized above, certain aspects of the present disclosure provide analysis cartridges for analyzing cells as well as non-cellular constituents of biological samples, such as blood. Also,certain aspects of the disclosure provide analysis cartridges that allow analysis of small volumes of biological samples.

[0046] In certain aspects, the disclosure provides an analysis cartridge for analyzing a biological sample, the analysis cartridge comprising:

[0047] a cellular analysis module for analyzing cells of the biological sample,

[0048] a chemical analysis module for analyzing non-cellular constituents of the biological sample, and

[0049] a filtration module that filters the biological sample by transferring the biological sample from the cellular analysis module through a filter to the chemical analysis module thereby delivering the non-cellular constituents of the biological sample to the chemical analysis module.

[0050] The term “biological sample” as used herein includes any sample comprising cells and non-cellular constituents. In some embodiments, a biological sample is a biological fluid sample.

[0051] A suitable biological sample analyzed in the analysis cartridges disclosed herein can be blood, urine, saliva, sweat, sputum, semen, mucus, lacrimal fluid, lymph fluid, amniotic fluid, interstitial fluid, lung lavage, cerebrospinal fluid, feces, or the like. A biological sample can also be a slurry of an otherwise solid tissue, such as a liver or a kidney, homogenized in a buffer to prepare a slurry comprising cells and non-cellular constituents. A biological sample can also comprise cultured cells in a suitable buffer. Additional examples of biological samples that could be analyzed in the analysis cartridges disclosed herein can be readily identified by a person of ordinary skill in the art and such embodiments are within the purview of the disclosure.

[0052] A portion of the biological sample from which the cells are removed is referenced herein as “a non-cellular portion of the biological sample.” A non-cellular portion of the biological sample comprises one or more non-cellular constituents, including metabolites and small molecules, such as glucose and other biochemicals as well as large biomolecules, such as proteins.

[0053] A chemical analysis module can analyze one or more non-cellular constituents.

[0054] A biological sample can be obtained from any suitable subject. “Subject” as used herein refers to any animal, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non-human. In some embodiments, the subject is a human.

[0055] Depending on the analyzed biological sample, different cells can be analyzed in the cellular analysis module. For example, if the biological sample is blood, the cellular analysis module is configured to analyze the blood for CBC. Additional embodiments of cells that couldbe analyzed in the cellular analysis module are well known to a person of ordinary skill in the art and such embodiments are within the purview of this disclosure.

[0056] Similarly, depending on the biological sample, different non-cellular constituents can be analyzed in the chemical analysis module. For example, if the biological sample is blood, the cellular analysis module is configured to analyze blood plasma for CMP. Additional embodiments of non-cellular constituents that could be analyzed in the chemical analysis module are well known to a person of ordinary skill in the art and such embodiments are within the purview of this disclosure.

[0057] In some cases, the filtration module comprises a first end fluidically connected to a sample inlet of the cellular analysis module and a second end fluidically connected to a sample inlet of the chemical analysis module, and a filter is disposed between the first end and the second end.

[0058] The term “fluidically connected” indicates that the two components of an analysis cartridge are directly or indirectly connected so that a fluid present in one component can be transferred to the other component. For example, a first end of a filtration module fluidically connected to a sample inlet of a cellular analysis module indicates that the sample inlet of the cellular analysis module can have a direct fluidic connection to the filtration module or intervening connections or chambers, such as a sample chamber or a diverter could be present between the sample inlet of the cellular analysis module and the filtration module.

[0059] In certain embodiments of an analysis cartridge, a cellular analysis module is stacked above a chemical analysis module. The term “stacked above” indicates that a module is physically positioned above another module. Thus, when a cellular analysis module is stacked above a chemical analysis module, the biological sample can flow from the cellular analysis module to the chemical analysis module under the force of gravity.

[0060] In certain such cases, the cellular analysis module is on a first substrate and the chemical analysis module is on a second substrate, the first substrate stacked above the second substrate. The first end of the filtration module is fluidically connected to the sample inlet of the cellular analysis module and the second end of the filtration module is fluidically connected to the sample inlet of the chemical analysis module. Also, a filter is disposed below the cellular analysis module such that a portion of a biological sample introduced into the cellular analysis module transfers under the force of gravity from the sample inlet of the cellular analysis module through the filter to the sample inlet of the chemical analysis module.

[0061] In certain such cases, a filtration module filters the biological sample as the biological sample transfers, under the force of gravity, from the cellular analysis module through the filter to the chemical analysis module.

[0062] In certain embodiments of an analysis cartridge, a chemical analysis module is stacked above a cellular analysis module. In certain such cases, the chemical analysis module is on a first substrate, the cellular analysis module is on a second substrate, wherein the first substrate is stacked above the second substrate. Also, the first end of the filtration module is fluidically connected to the sample inlet of the cellular analysis module and the second end of the filtration module is fluidically connected to the sample inlet of the chemical analysis module. A filter is disposed above the cellular analysis module. The filtration module filters the biological sample as the biological sample transfers, under a force that moves the biological fluid against the force of gravity, from the sample inlet of the cellular analysis module through the filter to the sample inlet of the chemical analysis module.

[0063] To move the biological sample against the force of gravity, the filtration module comprises a force generator that generates the force to move the biological sample against the force of gravity. The force generator can be a pump, such as a vacuum pump, a peristaltic pump, or a diaphragm pump. Additional examples of suitable pumps that could be used in the analysis cartridges disclosed herein are well known to a person of ordinary skill in the art and such embodiments are within the purview of the disclosure.

[0064] In certain other aspects, the disclosure provides an analysis cartridge for analyzing cells and non-cellular constituents of a biological sample, the analysis cartridge comprising:

[0065] a cellular analysis module for analyzing the cells of the biological sample,

[0066] a chemical analysis module for analyzing non-cellular constituents of the biological sample, and

[0067] wherein the cellular analysis and the chemical analysis module are in substantially the same horizontal plane and are connected by the filtration module.

[0068] In certain such cases, the filtration module comprises a first end fluidically connected to a sample inlet of the cellular analysis module and a second end fluidically connected to a sample inlet of the chemical analysis module, and a filter disposed between the first end and the second end. The filtration module filters the biological sample as the biological sample transfers from the sample inlet of the cellular analysis module through the filter to the sample inlet of the chemical analysis module. In some cases, a diverter can be present between the sample inlet of the cellular analysis module and the filtration module. In some cases, a hemoglobin detection chamber can be fluidically connected to sample inlet of a cellular analysis module such that a portion of the biological sample can be used to estimate hemoglobin in the biological sample.

[0069] To generate force to move the biological sample from the sample inlet of the cellular analysis module to the sample inlet of the chemical analysis module, the filtration module cancomprise a force generator. A force generator can be a pump, such as a vacuum pump, a peristaltic pump, or a diaphragm pump.Cellular analysis module

[0070] In certain embodiments, the analysis cartridge comprises a cellular analysis module. In some cases, a cellular analysis module comprises an imaging chamber comprising a top panel and a bottom panel separated by a suitable gap to produce a monolayer of cells between the top and the bottom panels. In some cases, the imaging chamber has a sample delivery edge, wherein the gap between the top panel and the bottom panel is accessible to a biological sample delivered to the sample delivery edge. The analysis cartridge can further comprise a sample loading port that delivers a biological sample to the sample delivery edge thereby allowing the biological sample to load into the imaging chamber.

[0071] Certain exemplary cellular analysis modules that could be used in the analysis cartridges disclosed herein are described in the United States Provisional Patent Application No. 63 / 634,315, which is incorporated herein by reference in its entirety.

[0072] An exemplary embodiment of a cellular analysis module used in the analysis cartridges disclosed herein is provided in FIG. 1. As shown in FIG. 1, the cellular analysis module 100 comprises a sample chamber or sample inlet 101, a sample deposition member (not shown) that delivers a biological sample into the sample chamber or sample inlet. The sample loading port 105 is fluidically connected to the sample chamber or sample inlet via the sampling tube 102. The cellular analysis module can comprise the pump 103 that can force the biological sample from the sample chamber or sample inlet to the sample loading port 105 via the sampling tube 102. The sample loading port can contain one or more reagents for treating the biological sample before it is loaded into the imaging chamber.

[0073] In some cases, the sample loading port contains one or more dyes to stain cells present in the biological sample. The dyes can be for specifically staining blood cells. Non-limiting examples of such dyes include one or more non-fluorescent or fluorescent dyes such as Eosin, Methylene Blue, Acridine Orange (also referred to as “Basic Orange 15” or “ACO”), or Astrazon Orange (also referred to as “AO” or Basic Orange 21), a component to bind to nuclear DNA in cells (e.g., blood cells such as WBCs).

[0074] In some embodiments, the imaging chamber can contain one or more dyes to stain cells present in a biological sample.

[0075] The one or more dyes in the imaging chamber can be spray dried into the imaging chamber. As the one or more dyes, such as acridine orange, is sprayed dried, the one or more dyes form a thin layer of coating in the imaging chamber. While the blood sample is drawn into the imagingchamber, for example, by capillary force, the one or more dyes dissolve in the sample and mix with it. In some cases, the chamber height is such that the chamber dimensions facilitate diffusion of the one or more dyes into the sample.

[0076] The one or more dyes in the imaging chamber can also be in a coating that releases the one or more dyes. Such coating can be a delayed-release coating. In some cases, the coating comprises one or more dyes and one or both of ethyl cellulose and hydroxypropyl methyl cellulose (HPMC). Other suitable materials can also be used for the coating and such alternatives are known in the art. In some cases, the coating of release control material and the one or more dyes in the imaging chamber is suitable for a roll-to-roll procedure.

[0077] In a specific embodiment, a coating comprises a cellulose film that can disintegrate in the presence of a sample, such as a blood sample. In certain such embodiment, the cellulose film comprises acridine orange and is prepared as shown below:

[0078] For a desired volume of the film to be prepared, add in a beaker water at a volume of one- third of the desired volume.

[0079] Raise the temperature to approximately 90°C.

[0080] Weigh Hydroxylpropyl Methyl Cellulose (HPMC) within 3 to 5% of the film solution.

[0081] Add HPMC to the warm water in the reaction beaker with constant stirring.

[0082] Add to the reaction beaker glycerin to 0.25% w / v of the film solution.

[0083] Add microcrystalline cellulose up to 0.8% w / v of the film solution.

[0084] Add to the reaction beaker with constant stirring the remaining two-third of the cold water.

[0085] Cast film or spin coat the solution on the surface of imaging chamber to form the film as the solvent evaporates.

[0086] Print acridine orange on the surface of the imaging chamber. In some cases, the concentration of acridine orange is 90 ng per microliter of blood sample.

[0087] For a delayed-release coating, the pore size, film porosity, and / or the formulation of specific one or more dyes can be appropriately selected to control the releasing of the one or more dyes and to obtain uniformity and efficiency of the staining of cells in a blood sample.

[0088] To facilitate mixing of one or more dyes with the biological sample, the sample loading port can contain a micro-mixer, such as Eddie-effect obstructor or Herringbone mixer.

[0089] The sample loading port is fluidically connected to the sample delivery edge 104 of the imaging chamber 106.

[0090] The sample loading port can deposit a biological sample into the imaging chamber. A sample loading port can deposit between 0.1 pl and 10 pl, such as 0.5 pl, 1 .0 pl, 1 .5 pl, 2.0 pl, 2.5 pl, 3.0 pl, 3.5 pl, 4.0 pl, 4.5 pl, 5.0 pl, 5.5 pl, 6.0 pl, 6.5 pl, 7.0 pl, 7.5 pl, 8.0 pl, 8.5 pl, 9.0 pl, 9.5 pl, or 10.0 pl of a biological sample into the imaging chamber.

[0091] The dimensions of the imaging chamber are adjusted according to the volume of the biological sample to be analyzed. Accordingly, the imaging chamber can have a gap between the top panel and the bottom panel between 1 pm and 50 pm, such as between 3 pm and 10 pm. For example, in some cases, such gap is between 1 pm and 100 pm, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pm. The imaging chamber can be a quadrilateral, such as a square or a rectangle. The imaging chamber can also be a circle, ellipse, triangle, or any other appropriate shape suitable for imaging a monolayer of cells. The surface area of the imaging chamber can be from 50 mm2to 1000 mm2, such as from 50 mm2to 200 mm2. A person of ordinary skill in the art can determined appropriate dimensions and / or the shape for the imaging chamber so that the imaging chamber can hold a desirable volume of a biological sample.

[0092] The sample inlet or sample chamber of a cellular analysis module is connected to a filtration module, which is described below in detail. The fluidic connection between the sample inlet or sample chamber and the filtration module can comprise a hemoglobin detection chamber (not shown). The hemoglobin detection chamber is configured to measure hemoglobin concentration of the biological sample, such as blood, loaded in the sample inlet or sample chamber. Such analysis of hemoglobin can be based on optical estimation of hemoglobin in the sample.

[0093] The imaging chamber, once loaded with a biological sample, can be imaged by a microscope. The images obtained can be saved digitally and analyzed visually or by using an appropriate computer program to provide analysis of the cells, for example, cell counts within the biological sample.

[0094] In some cases, the sample is a blood sample and the images of the sample obtained from the imaging chamber and microscope are analyzed to determine the numbers and / or concentrations of various types of cells in the blood to provide CBC analysis.Chemical analysis module

[0095] In certain embodiments, the analysis cartridge comprises a chemical analysis module.

[0096] In some cases, the chemical analysis module comprises a sample distribution channel that distributes a non-cellular portion of a biological sample into a plurality of detection chambers and the plurality of detection chambers detect a plurality of non-cellular constituents.

[0097] Certain such exemplary chemical analysis modules are described in the United States Provisional Patent Application Nos. 63 / 563,861 and 63 / 566,072, which are both incorporated herein by reference in their entireties.

[0098] In certain cases, the chemical analysis module comprises a top panel and a bottom panel and a plurality of channels disposed between the top panel and the bottom panel. The plurality ofchannels can run from a proximal end to a distal end of the top panel and the bottom panel. The plurality of channels are configured for analyzing a plurality of analytes. Certain such exemplary chemical analysis modules are described in the United States Provisional Patent Application No. 63 / 612,216, which is incorporated herein by reference in its entirety.

[0099] In certain embodiments, a chemical analysis module comprises a sample receiving channel connected to the filtration module for receiving a non-cellular portion of a biological sample. A sample so received can be mixed with a diluent and distributed via a sample distribution channel to a plurality of detection chambers that analyze a plurality of non-cellular constituents. An exemplary chemical analysis module is described in FIG. 2.

[0100] In the chemical analysis module of FIG. 2, the sample receiving channel 201 is fluidically connected, optionally though a valve 202, to a sample metering chamber 214. The sample metering chamber can be of specific dimensions to allow loading of a specific volume of a sample, for example, via capillary force or by a pump. Once filled, the sample metering chamber can be isolated from other channels in the chemical analysis module, for example, by closing valves 202 and 203, to capture a sample of a specific metered volume.

[0101] The chemical analysis module can further comprise a diluent blister 204, which provides diluent for mixing with the non-cellular portion of the biological sample. The diluent blister may be any suitable reservoir or container (e.g., having rigid or flexible walls) for holding a diluent fluid (e.g., a blood diluent fluid, such as saline). The volume of the diluent blister may vary, and can range in some instances from 5 pl to 1000 pl, such as 10 pl to 500 pl, and including 15 pl to 250 pl.

[0102] A diluent blister can be connected to a diluent metering chamber 205, optionally through the valve 206, to the diluent metering chamber. The diluent metering chamber can be of specific dimensions to allow loading of a specific volume of a diluent, for example, via capillary force or mediated by a pump. Once filled, the diluent metering chamber can be isolated from other channels in the chemical analysis module, for example, by closing valves 206, 207, and 208, to capture a diluent of a specific metered volume. The volume of the diluent metering chamber can be used to calculate the concentrations of the non-cellular constituents following dilution of the sample.

[0103] After a desired volume of non-cellular portion of a biological sample (herein, also referenced as “sample” for the purposes of analysis in a chemical analysis module) is loaded in the sample chamber and a desired volume of diluent is loaded in the diluent chamber, appropriate amounts of the sample and the diluent can be moved to the diluent mixing chamber, for example, by pumps 216 and 217 to drive the desired volumes of the sample and the diluent into the diluent mixing chamber 209.

[0104] Once a diluted sample is produced in the diluent mixing chamber, the diluted sample is ready for analysis. Accordingly, the diluted sample can be pushed through the sample distribution channel 210. The pump 211 can be used to push the diluted sample through the sample distribution channel.

[0105] The sample distribution channel 210 is connected to a plurality of detection chambers 212 via a plurality of detection chamber channels 213. Certain detection chambers may have a second channel with the capillary stop 218.

[0106] The plurality of detection chambers can be arranged in any suitable arrangement. For example, as shown in FIG. 2, the plurality of detection chambers can be arranged in a circular fashion around a circular sample distribution channel 210. Any other suitable arrangement can be chosen by a person of ordinary skill in the art and such embodiments are within the purview of the disclosure.

[0107] The plurality of detection chambers can contain reagents, for example, dried reagents, that are configured to analyze specific compounds within the non-cellular portion of the biological sample. In some cases, the cartridge is pre-filled with reagents and is ready to use. In some embodiments, all or a portion of the reagents may be present. Depending on the type of reagents present in the detection chambers and the resultant signal produced for the analyzed non-cellular constituents, appropriate detectors (not shown) are used to detect the signals.

[0108] The analysis cartridges may need to be stored in appropriate conditions to preserve the reactivity of the reagents. For example, depending on the reagents present, the analysis cartridges may need to be stored in a refrigerator or a freezer before use. When the reagents are not sensitive to room temperature, the analysis cartridges may be stored at room temperature.

[0109] The one or more dry reagents may in certain embodiments comprise a one or more non- fluorescent or fluorescent dyes such as Eosin, Methylene Blue, Acridine Orange (also referred to as "Basic Orange 15" or "ACO"), or Astrazon Orange (also referred to as "AO" or Basic Orange 21), a component to bind to nucleic acids, an anticoagulant, an antibody, an antibody fragment, an ionophore, an enzyme, a set of enzymes, a peptide with a cleavable detectable moiety, a substrate, an optical marker dye identifying a type of assay bead, and / or combinations thereof. In another case, when dry reagents are mixed with sample, for example, plasma, a colorimetric reaction occurs which may be optically detectable.

[0110] Analysis cartridges of the invention include a plurality of detection chambers. Detection chambers for use in the subject cartridges may be any microfluidic component configured for the analysis (e.g., optical analysis) of a non-cellular constituents of biological samples. Exemplary detection chambers include, but are not limited to, microcuvettes. The number of detection chambers in the plurality may vary. In some cases, the number of detection chambers in theplurality ranges from 2 to 100, such as 5 to 50, such as 10 to 25 and including 14 to 20. In some cases, cartridges include 10 or more detection chambers, such as 11 or more detection chambers, such as 12 or more detection chambers, such as 13 or more detection chambers, such as 14 or more detection chambers, such as 15 or more detection chambers, such as 16 or more detection chambers, such as 17 or more detection chambers, such as 18 or more detection chambers, such as 19 or more detection chambers, and including 20 or more detection chambers. The shape and size of the detection chambers in the plurality may vary, as desired. In certain cases, the detection chambers of the plurality have an elongated structure (e.g., having a length greater than width). The elongate structure may have any convenient cross-sectional shape, where cross-sectional shapes of interest include, but are not limited to rectilinear cross-sectional shapes, e.g., squares, rectangles, trapezoids, triangles, hexagons, etc., curvilinear cross-sectional shapes, e.g., circles, ovals, as well as irregular shapes, e.g., a parabolic bottom portion coupled to a planar top portion. In certain cases, the detection chambers of the plurality have a circular cross section. In other embodiments, the detection chambers of the plurality have a square cross section. In still other embodiments, detection chambers of the plurality have a rectangular cross section. The volume of the detection chambers may also vary. In some cases, detection chambers of the plurality have a volume ranging from 0.3 pl to 500 pl, such as 2 pl to 300 pl, such as 3 pl to 200 pl, such as 4 pl to 100 pl and including 5 pl to 10 pl. In select versions, detection chambers of the plurality have a volume ranging from 0.3 pl to 50 pl. In certain cases, detection chambers of the plurality have a volume of 5 pl or more, such as 6 pl or more, such as 7 pl or more, such as 8 pl or more, such as 9 pl or more, and including 10 pl or more. In some instances, detection chambers of the plurality have a diameter ranging from 0.1 mm to 20 mm, such as 0.5 mm to 15 mm, such as 1 mm to 10 mm, and including 1.5 mm to 2 mm. In certain cases, detection chambers of the plurality have a diameter of 1.5 mm or more, such as 1.6 mm or more, such as 1.7 mm or more, such as 1.8 mm or more, such as 1.9 mm or more, and including 2 mm or more. Adjacent detection chambers of the plurality may be separated by a distance ranging from 1 mm to 10 mm, such as 2 mm to 8 mm, and including 4 mm to 5 mm. Space between detection chambers may in some cases be sufficient to ensure that each one can be interrogated by a beam of light from the illuminator without interfering with its neighbor detection chambers. The detection chambers of the plurality may be arranged in any suitable pattern. In some cases, the detection chamber array is arranged in a staggered pattern. In other cases, the detection chamber array is arranged in a concentric pattern. The detection chambers in the subject cartridge may be constructed from any suitable material. In some cases, the detection chambers are comprised of a polymeric material that is transparent in a detection wavelength band. In some such cases, the detection chambers of the plurality are comprised of polystyrene (PS), PMMA, CoC, or CoP.

[0111] Detection chambers of the subject analysis cartridges may include an inlet for receiving a diluted sample, and an outlet where air and / or excess diluted sample fluid may escape as the detection chambers are being filled. In some embodiments where the detection chambers of the plurality are comprised of an elongate structure, the detection chambers include an inlet at a proximal end of the elongate structure, and an outlet at the distal end of the elongate structure. In certain instances, the cartridge is configured such that the detection chambers are arranged upright (i.e., vertically). In some such embodiments, the inlets may be arranged at the bottom such that the detection chambers fill with diluted sample from the bottom and ascend via capillary action. In some cases, this arrangement is sufficient to minimize the generation of air bubbles when sample fluid fills the detection chambers. In some alternate embodiments, detection chambers fill from the top-down. In certain cases, the detection chambers are light- accessible at certain windows. In some such cases, each detection chamber in the plurality comprises a first light- accessible window configured to permit entry of light, and a second light-accessible window configured to permit an exit of the light from the light source. The remainder of the detection chambers may or may not also be light-accessible. In select versions, aside from the first and second light- accessible windows, the detection chambers are opaque.

[0112] In embodiments, the outlets of the detection chambers have edges that are not perpendicular to the edges of the detection chamber. In some such embodiments, the outlets are beveled. Put another way, at least a portion of the outlet may be chamfered. Beveling the interface between the detection chamber and additional components (e.g., outlet vent, overflow reservoir, etc.) may be helpful to reduce bubble formation.

[0113] In some embodiments, cartridges include one or more capillary stops (i.e., capillary stop valves). As is understood in the art, capillary stops halt the flow of liquid in microchannels without external intervention using an abrupt change in microchannel geometry. In some cases, the capillary stop constitutes an abrupt expansion of detection chamber geometry. In other cases, the capillary stop constitutes an abrupt narrowing of detection chamber geometry. In certain instances, microfluidic channels are molded between detection chambers for transferring sample fluid and waste. In select instances, cartridges include an overflow reservoir fluidically connected to each detection chamber in the plurality. Said overflow reservoir may constitute a void in the cartridge into which diluted sample fluid may flow if / when said fluid surpasses the capillary stop. Additionally or alternatively, cartridges may include an overflow outlet vent gaseously connected to each capillary stop. Said overflow outlet vent may constitute a microfluidic channel gaseously connected to each capillary stop that vents to the surrounding environment.Filtration Module

[0114] In some embodiments, the analysis cartridges described herein comprise a filtration module. A filtration module is fluidically connected at the one end to a cellular analysis module and at the other end to a chemical analysis module. For example, a filtration module is fluidically connected at the one end to a sample inlet or sample chamber of a cellular analysis module and at the other end to a sample inlet or sample chamber of a chemical analysis module. A filtration module can comprise a filter for filtering cells from a biological sample and provide a non-cellular portion of the biological sample to a chemical analysis module.

[0115] Any suitable filter can be used in a filtration module. A suitable filter is selected based on the type of biological sample to be analyzed, particularly, the type and size of cells to be filtered. For example, of the biological sample tested is blood, filter in a filtration module is selected such that it removes all types of blood cells from the blood and provides plasma or serum to a chemical analysis module. The pore size for separation of blood cells from a blood sample to filter out plasma is between 0.1 and 5 pm, such as 0.1 pm, 0.2 pm, 0.3 pm, 0.4 pm, 0.5 pm, 0.6 pm, 0.7 pm, 0.8 pm, 0.9 pm, 1 pm, 2 pm, 3 pm, 4 pm, or 5 pm. Depending on the biological sample to be filtered, a person of ordinary skill in the art can determine appropriate pore size for a filter in a filtration module.

[0116] In certain aspects, a filtration module filters the biological sample as it passes through the filter under the force of gravity. In some other cases, a filtration module filters the biological sample as it passes through the filter under a force exerted via a pump, a vacuum, or capillary action.

[0117] In some cases, the disclosure provides an analysis cartridge, comprising:

[0118] a cellular analysis module and a chemical analysis module, wherein the cellular analysis module is stacked above the chemical analysis module or the chemical analysis module is stacked above the cellular analysis module.

[0119] In some cases, the cellular analysis module is stacked above the chemical analysis module. In certain such cases, the filtration module is fluidically connected at the one end to the cellular analysis module, for example, to the sample inlet or sample chamber of the cellular analysis module and at the other end to the chemical analysis module, for example, to sample inlet or sample chamber of the chemical analysis module. A filter is disposed between the first end and the second end of the filtration module and filters a biological sample as it passes from the cellular analysis module to the chemical analysis module.

[0120] FIG. 3 provides a top view of the analysis cartridge 300 having a cellular analysis module 301 stacked above the chemical analysis module 302. The cellular analysis module 301 obstructs the view of the chemical analysis module 302 and, therefore, the chemical analysis module is onlypartially visible. The filtration module 303 connects the cellular analysis module to the chemical analysis module stacked below it.

[0121] FIG. 4 provides a side view of the analysis cartridge 400, which is similar to the analysis cartridge 300 shown in FIG. 3. The cellular analysis module 401 is connected to the chemical analysis module 402 located below it. The filtration module 403 connects the cellular analysis module with the chemical analysis module. The blood sample 404 is loaded onto the sample inlet 405 of the cellular analysis module. The portion 406 of the loaded blood sample is analyzed in the cellular analysis module. Another portion 407 of the loaded blood sample flows down under the force of gravity through the filter 408. The filter 408 retains blood cells 409 while allowing the blood plasma 410 to drain into the sample inlet 411 of the chemical analysis module.

[0122] In some cases, the cellular analysis module and the chemical analysis module are rotatable around the axis that runs perpendicular to the filtration module. For example, in the cartridge 300 shown in FIG. 3, the cellular analysis module can rotate clockwise and / or counterclockwise around the axis that runs perpendicular to the plane of the cellular analysis module and through the filtration module.

[0123] Alternatively, the chemical analysis module is stacked above the cellular analysis module. In certain such cases, the filtration module is fluidically connected at the one end to the cellular analysis module, for example, through a sample inlet or sample chamber of the cellular analysis module and at the other end to the chemical analysis module, for example, to sample inlet or sample chamber of the chemical analysis module that is located above the cellular analysis module. The filtration module filters a biological sample as it passes, under a force that moves the biological sample against the force of gravity, from the cellular analysis module to the chemical analysis module.

[0124] The filtration module can have a membrane or filter for filtering cells from the non-cellular portion of the biological sample. When the cellular analysis module is stacked above the chemical analysis module, a biological sample, such as blood can transfer under the force of gravity as it is filtered to produce non-cellular constituents of the biological sample, such as plasma. When the chemical analysis module is stacked above the cellular analysis module, a biological sample can be pushed upwards through the filter, for example, via a pump or vacuum, as it is filtered to produce non-cellular constituents of the biological sample.

[0125] In certain aspects, the disclosure provides an analysis cartridge, comprising:

[0126] a cellular analysis module and a chemical analysis module, wherein the cellular analysis module and the chemical analysis module are in substantially the same horizontal plane.

[0127] In some cases, the cellular analysis module and the chemical analysis module are connected via a filtration module.

[0128] In certain such cases, the filtration module is fluidically connected, for example, to a sample inlet and / or a sample chamber of a cellular analysis module and is configured to receive a biological sample from the cellular analysis module. The filtration module filters the biological sample as it transfers from the cellular analysis module to the chemical analysis module thereby delivering the non-cellular portion of the biological sample to the chemical analysis module.

[0129] FIG. 5 describes an exemplary analysis cartridge 500 having the cellular analysis module 501 and the chemical analysis module 502 that are in substantially the same horizontal plane. The cellular analysis module 501 can have various components as described elsewhere in this disclosure, for example, FIG. 1 and its relevant description. Similarly, the chemical analysis module 502 can have various components as described elsewhere in this disclosure, for example, FIG. 2 and the relevant description. These two modules are connected via the filtration module 503.

[0130] The filtration module can comprise a membrane or filter and fluidic connections to the cellular analysis module and the chemical analysis module.

[0131] In certain such cases, the biological sample is forced through the filter under the force exerted by a pump or vacuum.

[0132] Thus, in the exemplary cartridge shown in FIG. 5, a biological sample can be introduced in the sample inlet or sample port 505 of the cellular analysis module 501. The pump 506 can force a portion of the sample to the sample loading port 507 and another portion of the sample to the filter 508. Once through the filter 508 the non-cellular portion of the biological sample is delivered to the sample metering chamber 509.Methods

[0133] The analysis cartridges disclosed herein can be used for analyzing a biological sample, for example, a blood sample for its cellular as well as non-cellular constituents.

[0134] A suitable biological sample that can be analyzed in the methods disclosed herein is identified elsewhere in this disclosure and methods of analyzing such samples are within the purview of the disclosure.

[0135] Any structural elements of the analysis cartridges described elsewhere in this disclosure, for example, those described under “Analysis Cartridges” above, are applicable to the methods disclosed herein. For example, the structures of “cellular analysis modules” and “chemical analysis modules” as discussed elsewhere in this disclosure also applicable to the methods disclosed herein.

[0136] Certain aspects of the disclosure provide a method of analyzing a biological sample, for example, a blood sample, for its cellular as well as non-cellular portion, such as plasma, in the analysis cartridges disclosed herein.

[0137] In some cases, the methods comprise delivering to a sample inlet or sample chamber of a Cellular analysis module a suitable amount of a biological sample. From the delivered biological sample, a small volume, for example, between 0.1 pl and 2 pl, such as 0.1 pl, 0.2 pl, 0.3 pl, 0.4 pl, 0.5 pl, 0.6 pl, 0.7 pl, 0.8 pl, 0.9 pl, 1.0 pl, 1.1 pl, 1.2 pl, 1.3 pl, 1.4 pl, 1.5 pl, 1.6 pl, 1.7 pl, 1.8 pl, 1.9 pl, or 2.0 pl is delivered to an imaging chamber of the Cellular analysis module. Also, a portion of the sample delivered to the sample inlet or sample chamber of a Cellular analysis module, a suitable amount is filtered through the filtration module to deliver an amount of non- cellular constituents of the biological sample to the Chemical analysis module. For example, a non-cellular constituent of a biological sample in a volume ranging from 2 pl to 500 pl, such as 3 pl to 300 pl, such as 5 pl to 200 pl, such as 8 pl to 100 pl, such as 9 pl to 50 pl and including 10 pl to 40 pl can be delivered to the sample inlet or sample chamber of a Chemical analysis module.

[0138] A biological sample containing cells, such as a blood sample loaded into the imaging chamber can be analyzed to obtain, store, and process images of the biological sample. Such images can be used to determine cellular composition of the biological sample. For example, depending on the detection of certain types and / or number of blood cells in the images obtained from the imaging chamber, the methods comprise providing CBC values for the blood sample.

[0139] A biological sample containing non-cellular constituents, such as plasma sample loaded into the Chemical analysis module can be analyzed to obtain concentrations of one or more biomolecules.

[0140] In some embodiments, methods disclosed herein comprise assaying clinical chemistry panels in a blood sample. The clinical chemistry panels refer to groups of tests that are routinely ordered to determine a subject’s general health status. In some cases, the clinical chemistry panels include metabolic panels. The clinical chemistry panels help evaluate, for example, the body's electrolyte balance and / or the status of several major body organs. In some cases, the assays are performed on a blood sample, usually drawn from a vein. Examples of clinical chemistry panels that may be detected by assays of the present disclosure include, but are not limited to, basic metabolic panel (BMP), comprehensive metabolic panel (CMP), electrolyte panel, lipid panel, liver panel, renal panel, and thyroid function panel. The basic metabolic panel (BMP) includes 8 tests, all of which are found in the CMP. The BMP provides information about the current health of kidneys and respiratory system as well as electrolyte and acid / base balance and level of blood glucose. The CMP measurement is used for liver and kidney health, level of blood glucose, acid / base balance in blood, fluid and electrolyte balance, and important blood proteins. In somecases, the CMP measures glucose, calcium, total amount of albumin and globulins, bilirubin, BUN (blood urea nitrogen), creatinine, albumin, sodium, potassium, bicarbonate, chloride, alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate aminotransferase (AST). The electrolyte panel is used to detect a problem with the body’s fluid and electrolyte balance. For example, the electrolyte panel measures the blood levels of carbon dioxide, chloride, potassium, and sodium. The lipid panel is used to assess a subject’ s risk of developing cardiovascular disease. For example, the lipid panel measures the amount of cholesterol and other fats in blood, such as total cholesterol, LDL (low-density lipoprotein), HDL (high-density lipoprotein), and triglycerides. The liver panel (hepatic function panel) is used to screen for, detect, evaluate, and monitor acute and chronic liver inflammation (hepatitis), liver disease and / or damage. The liver panel measures different enzymes, proteins, and other substances made by liver. For example, the liver panel includes albumin, total protein, ALP, ALT, AST, gamma-glutamyl transferase (GGT), bilirubin, Lactate dehydrogenase (LD), Prothrombin time (PT). The renal panel (kidney function panel) includes tests such as albumin, creatinine, BUN, eGFR to evaluate kidney function. The thyroid Function Panel is used to evaluate thyroid gland function and to help diagnose thyroid disorders. The thyroid function panel measure thyroid hormone such as thyroxine (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH). In some cases, a high TSH level indicates that the thyroid gland is not making enough thyroid hormone (primary hypothyroidism). The opposite situation, in which the TSH level is low, usually indicates that the thyroid is producing too much thyroid hormone (hyperthyroidism). In other cases, the finding of an elevated TSH and low free T4 (FT4) or free T4 index (FTI) indicates primary hypothyroidism due to disease in the thyroid gland. A low TSH and low FT4 or FTI indicate hypothyroidism due to a problem involving the pituitary gland. A low TSH with an elevated FT4 or FTI is found in individuals who have hyperthyroidism. These clinical chemistry panels are well known in the art and are further described in the assay portion of the present disclosure.

[0141] In some cases, such as where the cartridge is employed for a complete metabolic panel (CMP), reagents comprise one or more of the following: 2,4,6-Tribromo-3-hydroxybenzoic acid (TBHBA), 2-Chloro-4-nitrophenyl-a-maltotrioside (CNPG3), 2-Methyl-4-isothizolin-3-one hydrochloride (MIT), 4,7,13,16,21-Pentaoxa-l,10-diazabicyclo[8.8.5]tricosane (Kryptofix 221), 4-Aminoantipyrine hydrochloride, Adenosine 5 ’ -diphosphate, Adenosine 5’ -triphosphate, a- ketoglutaric acid, Amylase, Arsenazo III, sodium salt, Ascorbate oxidase (Ciicurbita spp.), Bilirubin oxidase, Bromcresol purple, Calcium acetate, Creatine amidinohydrolase (Actinobacillus spp.), Creatinine amidohydrolase (Pseudomonas spp.), Cupric sulfate, Ethylene glycol-bis(B-aminoethyl ether)-N,N,N’,N’ -tetraacetic acid (EGTA), Ethylenediaminetetraaceticacid (EDTA), B-Galactosidase, Glucose-6-phosphate dehydrogenase (yeast), Glutamate Dehydrogenase (bovine liver), Glutamine synthetase, Hexokinase (yeast), Imidazole, Lactate dehydrogenase, L-alanine, L-aspartic acid, L-glutamic acid, Magnesium chloride, Magnesium sulfate, Malate dehydrogenase (porcine heart), Manganese chloride, N-Acetyl cysteine, B- Nicotinamide adenine dinucleotide (NAD), B-Nicotinamide adenine dinucleotide, reduced (NADH), o-Nitrophenyl-B-D-galactopyranoside (ONPG), Peroxidase (horseradish), Phosphoenol pyruvate, Phosphoenol pyruvate carboxylase, p-NPP, Potassium ferrocyanide, Potassium iodide, Pyruvate kinase, Sarcosine oxidase (microorganism), Sodium potassium tartrate, Urease (jack bean), Zinc sulfate, as well as other buffers, surfactants, excipients and preservatives.

[0142] In some cases, it may be desirable to analyze a control sample. The control sample may be analyzed concurrently with the sample from the subject as described above. The results obtained from the subject sample can be compared to the results obtained from the control sample. Standard curves may be provided, with which assay results for the sample may be compared. Using samples taken from multiple donors, standard curves can be provided for reference levels in normal healthy subjects.

[0143] In some cases, the method comprises introducing the biological sample into the sample inlet of a cellular analysis module and causing a portion of the biological sample delivered to the imaging chamber of the cellular analysis module. Further steps of the method can comprise causing a portion of the biological sample to be transferred to the filtration module to be filtered and introduced into the sample inlet of the chemical analysis module.

[0144] In some cases, the methods comprise, delivering a biological sample introduced into the sample inlet of the cellular analysis module to the sample imaging chamber of the cellular analysis module. Such delivering may comprise actuating a force generator, such as a pump, to transfer the biological sample to the sample loading port, in which the biological sample can be mixed with dyes or other reagents, optionally, using a micro-mixer, such as Eddie-effect obstructor or Herringbone mixer disposed in the sample loading port.

[0145] After a biological sample is loaded into the imaging chamber, the methods further comprise obtaining images of the cells in the loaded sample and analyzing the images to estimate the types and / or numbers of the cells present in the biological sample.

[0146] Similarly, after a biological sample is loaded into the chemical analysis module, the method comprises causing the sample to be loaded into a sample metering chamber. Causing the sample to be loaded into a sample metering chamber can comprise actuating a pump that forces the sample from the sample inlet into the sample metering chamber.

[0147] To dilute the sample in an appropriate diluent, the method can further comprise causing a diluent from a diluent blister to be loaded into a diluent metering chamber. Causing the diluentto be loaded into a diluent metering chamber can comprise actuating a force generator, such as a pump that forces the diluent from the diluent blister into the diluent metering chamber.

[0148] Further steps of the methods can comprise mixing an appropriate amount of the diluent with an appropriate amount of the sample to produce a diluted sample. Such dilution can be performed by actuating a force generator, such as a sample pump that pushes the sample from the sample metering chamber into a diluent mixing chamber and actuating another force generator, such as a diluent pump that pushes the diluent from the diluent metering chamber into the diluent mixing chamber. Thus, the sample is mixed with the diluent in the diluent mixing chamber.

[0149] The diluted sample can then be distributed to the number of detection chambers. Such distributing can be achieved by actuating a sample distribution pump that distributes the sample into the sample distribution channel, thereby filling a plurality of detection chambers with the diluted sample.

[0150] To detect and / or quantify a plurality of non-cellular constituents of the sample, the method further comprises quantifying signals from the plurality of detection chambers and detecting and / or quantifying the plurality of non-cellular constituents. Such detecting the signals can comprise detecting one or more optical signals, one or more electrochemical signals, one or more chemical signals, and the like. Appropriate sensors can be implemented for detecting and / or quantifying the signals.

[0151] Thus, in some embodiments, the methods of analyzing a biological sample comprise:

[0152] introducing the biological sample into the sample inlet of the cellular analysis module,

[0153] transferring the biological sample to the sample loading port and delivering the biological sample into the imaging chamber of the cellular analysis module,

[0154] obtaining images of the cells in the loaded sample and analyzing the images to estimate the types and / or numbers of the cells present in the biological sample,

[0155] transferring a portion of the biological sample introduced into the sample inlet of the cellular analysis module to the filtration module,

[0156] filtering the portion of the biological sample transferred to the filtration module,

[0157] introducing the filtered biological sample into the sample inlet of a chemical analysis module,

[0158] loading the sample into a sample metering chamber,

[0159] loading a diluent from a diluent blister into a diluent metering chamber,

[0160] mixing in a diluent mixing chamber an appropriate amount of the diluent with an appropriate amount of the sample to produce a diluted sample,

[0161] distributing the diluted sample into the number of detection chambers via a sample distribution channel thereby filling a plurality of detection chambers with the diluted sample,

[0162] quantifying signals from the plurality of detection chambers and detecting and / or quantifying the plurality of non-cellular constituents.

[0163] In the method described above, various steps, such as introducing, transferring, filtering, loading, and distributing can be performed by actuating or causing to actuate, for example, via an analyzer, one or more force generators, such as pumps to induce the desired movement of the biological sample, diluent, or diluted sample within the analysis cartridge.

[0164] Thus, in view of the above, a method for determining the presence and / or amount of cells and / or non-cellular constituents in a biological sample is provided. In some cases, the method comprises assaying the biological sample for cells and non-cellular constituents and comparing it to a control. In some cases, a calibrator is optionally, and is preferably, part of a series of calibrators in which each of the calibrators differs from the other calibrators in the series by the concentration of the analyte.Analyzers for analysis cartridges

[0165] In some aspects, the analysis cartridges are processed in an analyzer that contains software to execute one or more tasks, including the performance of the methods described herein. In some aspects, the analyzer contains software to automatically determine the next appropriate step in methods as described herein. For example, the instrument may contain software that determines amount or presence of an analyte of interest. The software may display this determination, such as on a graphical user interface.

[0166] In some aspects, the analyzer stores software that instructs a processor to execute a given task. In some aspects, the software stores machine readable instructions that instruct a processor to execute a given task. The machine-readable instructions may be one or more executable programs or portion(s) of an executable program for execution by a computer. The programs may be embodied in software stored on a non-transitory computer readable storage medium such as a CD-ROM, a floppy disk, a hard drive, a DVD, a Blu-ray disk, or a memory associated with the processors. Alternatively, the entire programs and / or parts thereof could alternatively be executed by a device other than the processors and / or embodied in firmware or dedicated hardware. Additionally or alternatively, processes may be implemented by one or more hardware circuits (e.g., discrete and / or integrated analog and / or digital circuitry, an FPGA, an ASIC, a comparator, an operational- amplifier (op-amp), a logic circuit, etc.) structured to perform the corresponding operation without executing software or firmware.

[0167] The machine-readable instructions may be stored in one or more of a compressed format, an encrypted format, a fragmented format, a compiled format, an executable format, a packaged format, etc. Machine readable instructions as described herein may be stored as data (e.g., portionsof instructions, code, representations of code, etc.) that may be utilized to create, manufacture, and / or produce machine executable instructions. For example, the machine-readable instructions may be fragmented and stored on one or more storage devices and / or computing devices (e.g., servers). The machine-readable instructions may require one or more of installation, modification, adaptation, updating, combining, supplementing, configuring, decryption, decompression, unpacking, distribution, reassignment, compilation, etc. in order to make them directly readable, interpretable, and / or executable by a computing device and / or other machine. For example, the machine-readable instructions may be stored in multiple parts, which are individually compressed, encrypted, and stored on separate computing devices, wherein the parts when decrypted, decompressed, and combined form a set of executable instructions that implement a program such as that described herein.

[0168] In another example, the machine-readable instructions may be stored in a state in which they may be read by a computer, but require addition of a library (e.g., a dynamic link library (DLL)), a software development kit (SDK), an application programming interface (API), etc. in order to execute the instructions on a particular computing device or other device. In another example, the machine-readable instructions may need to be configured (e.g., settings stored, data input, network addresses recorded, etc.) before the machine-readable instructions and / or the corresponding program(s) can be executed in whole or in part. Thus, the disclosed machine- readable instructions and / or corresponding program(s) are intended to encompass such machine- readable instructions and / or program(s) regardless of the particular format or state of the machine- readable instructions and / or program(s) when stored or otherwise at rest or in transit.

[0169] The machine-readable instructions described herein can be represented by any past, present, or future instruction language, scripting language, programming language, etc. For example, the machine-readable instructions may be represented using any of the following languages: C, C++, Java, C#, Perl, Python, JavaScript, HyperText Markup Language (HTML), Structured Query Language (SQL), Swift, etc.

[0170] The machine readable instructions may be stored on a non-transitory computer and / or machine readable medium such as a hard disk drive, a flash memory, a read-only memory, a compact disk, a digital versatile disk, a cache, a random-access memory and / or any other storage device or storage disk in which information is stored for any duration (e.g., for extended time periods, permanently, for brief instances, for temporarily buffering, and / or for caching of the information). As used herein, the term non-transitory computer readable medium is expressly defined to include any type of computer readable storage device and / or storage disk and to exclude propagating signals and to exclude transmission media.

[0171] Thus, in some cases, an analyzer for analyzing a biological sample causes a biological sample to be introduced into the sample inlet of a cellular analysis module and transfer a portion of the biological sample into an imaging chamber.

[0172] In some cases, the analyzer can also cause the biological sample to be mixed with dyes or other reagents, optionally, using a micro-mixer, such as Eddie-effect obstructor or Herringbone mixer disposed in the sample loading port. The analyzer can then cause obtaining images of the cells in the loaded sample and analyzing the images to estimate the types and / or numbers of the cells present in the biological sample.

[0173] The analyzer can further cause a portion of the biological sample to be transferred to the filtration module to be filtered and introduce the filtered biological sample into the sample inlet of the chemical analysis module.

[0174] After a biological sample is loaded into the chemical analysis module, the analyzer can cause the sample to be loaded into a sample metering chamber. Causing the sample to be loaded into a sample metering chamber can comprise actuating a pump that forces the sample from the sample inlet into the sample metering chamber.

[0175] The analyzer can then cause the sample to be diluted in an appropriate diluent by causing a diluent from a diluent blister to be loaded into a diluent metering chamber. Causing the diluent to be loaded into a diluent metering chamber can comprise actuating a pump that forces the diluent from the diluent blister into the diluent metering chamber.

[0176] The analyzer can further cause mixing an appropriate amount of the diluent with an appropriate amount of the sample to produce a diluted sample. Such dilution can be caused by actuating a sample pump that pushes the sample from the sample metering chamber into a diluent mixing chamber and a diluent pump that pushes the diluent from the diluent metering chamber into the diluent mixing chamber. Thus, the analyzer causes the sample to be mixed with the diluent in the diluent mixing chamber.

[0177] Furthermore, the analyzer can cause the diluted sample to be distributed to the number of detection chambers. Such distributing can be caused by actuating a sample distribution pump that distributes the sample into the sample distribution channel, thereby filling a plurality of detection chambers with the diluted sample.

[0178] To detect and / or quantify a plurality of non-cellular constituents of the sample, the analyzer can further quantify signals from the plurality of detection chambers and detect and / or quantify the plurality of non-cellular constituents. Such detecting the signals can comprise detecting one or more optical signals, one or more electrochemical signals, one or more chemical signals, and the like.

[0179] Thus, in some embodiments, the disclosure discloses an analyzer comprising a non- transitory storage media comprising instructions, which when executed by a computer, cause the analyzer to:

[0180] introduce the biological sample into the sample inlet of a cellular analysis module,

[0181] transfer the biological sample to the sample loading port and deliver the biological sample into an imaging chamber of a cellular analysis module,

[0182] obtain images of the cells in the loaded sample and analyze the images to estimate the types and / or numbers of the cells present in the biological sample,

[0183] transfer a portion of the biological sample introduced into the sample inlet of the cellular analysis module to the filtration module,

[0184] filter the portion of the biological sample transferred to the filtration module,

[0185] introduce the filtered biological sample into the sample inlet of a chemical analysis module,

[0186] load the sample into a sample metering chamber,

[0187] load a diluent from a diluent blister into a diluent metering chamber,

[0188] mix in a diluent mixing chamber an appropriate amount of the diluent with an appropriate amount of the sample to produce a diluted sample,

[0189] distribute the diluted sample into the number of detection chambers via a sample distribution channel thereby filling a plurality of detection chambers with the diluted sample,

[0190] quantify signals from the plurality of detection chambers and detecting and / or quantifying the plurality of non-cellular constituents.

[0191] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0192] Clause 1. An analysis cartridge for analyzing a biological sample, the analysis cartridge comprising:

[0193] a cellular analysis module for analyzing cells of the biological sample,

[0194] a chemical analysis module for analyzing non-cellular constituents of the biological sample, and

[0195] a filtration module that filters the biological sample by transferring the biological sample from the cellular analysis module through a filter to the chemical analysis module thereby delivering the non-cellular constituents of the biological sample to the chemical analysis module.

[0196] Clause 2. The analysis cartridge of Clause 1 , wherein the filtration module comprises a first end fluidically connected to a sample inlet of the cellular analysis module and a second end fluidically connected to a sample inlet of the chemical analysis module, and a filter disposed between the first end and the second end.

[0197] Clause 3. The analysis cartridge of Clause 1 or 2, wherein the cellular analysis module is stacked above the chemical analysis module.

[0198] Clause 4. The analysis cartridge of Clause 3, wherein:

[0199] the cellular analysis module is on a first substrate, the chemical analysis module is on a second substrate, the first substrate stacked above the second substrate, and

[0200] the first end of the filtration module is fluidically connected to the sample inlet of the cellular analysis module and the second end of the filtration module is fluidically connected to the sample inlet of the chemical analysis module, and the filter is disposed below the cellular analysis module, and wherein the filtration module filters the biological sample as the biological sample transfers, under the force of gravity, from the sample inlet of the cellular analysis module through the filter to the sample inlet of the chemical analysis module.

[0201] Clause 5. The analysis cartridge of Clause 1 or 2, wherein the chemical analysis module is stacked above the cellular analysis module.

[0202] Clause 6. The analysis cartridge of Clause 5, wherein:

[0203] the chemical analysis module is on a third substrate, the cellular analysis module is on a fourth substrate, the third substrate stacked above the fourth substrate, and

[0204] the first end of the filtration module is fluidically connected to the sample inlet of the cellular analysis module and the second end of the filtration module is fluidically connected to the sample inlet of the chemical analysis module, and the filter is disposed above the cellular analysis module, and wherein the filtration module filters the biological sample as the biological sample transfers, under a force that moves the biological fluid against the force of gravity, from the sample inlet of the cellular analysis module through the filter to the sample inlet of the chemical analysis module.

[0205] Clause 7. The analysis cartridge of Clause 6, wherein the filtration module further comprises a force generator that generates the force to move the biological sample against the force of gravity.

[0206] Clause 8. The analysis cartridge of Clause 7, wherein the force generator is a pump.

[0207] Clause 9. The analysis cartridge of Clause 8, wherein the pump is a vacuum pump, a peristaltic pump, or a diaphragm pump.

[0208] Clause 10. The analysis cartridge of Clause 1 or 2, wherein the cellular analysis and the chemical analysis module are in substantially the same horizontal plane and are connected by the filtration module.

[0209] Clause 1 1 . The analysis cartridge of Clause 10, wherein the filtration module comprises a first end fluidically connected to a sample inlet of the cellular analysis module and asecond end fluidically connected to a sample inlet of the chemical analysis module, and a filter disposed between the first end and the second end.

[0210] Clause 12. The analysis cartridge of Clause 11, wherein the first end of the filtration module is fluidically connected to the sample inlet of the cellular analysis module and the second end of the filtration module is fluidically connected to the sample inlet of the chemical analysis module, and wherein the filtration module filters the biological sample as the biological sample transfers from the sample inlet of the cellular analysis module through the filter to the sample inlet of the chemical analysis module.

[0211] Clause 13. The analysis cartridge of Clause 12, wherein the filtration module further comprises a force generator that generates the force to move the biological sample from the sample inlet of the cellular analysis module to the sample inlet of the chemical analysis module.

[0212] Clause 14. The analysis cartridge of Clause 13, wherein the force generator is a pump.

[0213] Clause 15. The analysis cartridge of Clause 14, wherein the pump is a vacuum pump, a peristaltic pump, or a diaphragm pump.

[0214] Clause 16. The analysis cartridge of any one of Clauses 1 to 15, wherein the fluidic connection between the sample inlet of the cellular analysis module and the filtration module comprises a diverter that diverts a portion of the biological sample introduced into the sample inlet of the cellular analysis module to the filtration module.

[0215] Clause 17. The analysis cartridge of any one of Clauses 1 to 16, wherein the filtration module comprises a membrane filter that filters cells from the non-cellular portion of the biological sample.

[0216] Clause 18. The analysis cartridge of Clause 16, further comprising a hemoglobin detection chamber fluidically connected to the diverter.

[0217] Clause 19. The analysis cartridge of any one of Clauses 1 to 18, wherein the cellular analysis module comprises an imaging chamber comprising a top panel and a bottom panel separated by a suitable distance to produce a monolayer of cells between the panels.

[0218] Clause 20. The analysis cartridge of Clause 19, wherein the imaging chamber comprises:

[0219] a sample delivery edge where the gap between the top panel and the bottom panel is accessible to the biological sample delivered to the sample delivery edge, and

[0220] a sample loading port delivers the biological sample to the sample delivery edge thereby allowing the biological sample to fill the imaging chamber.

[0221] Clause 21. The analysis cartridge of Clause 20, wherein the sample loading port contains one or more dyes that stain cells present in the biological sample.

[0222] Clause 22. The analysis cartridge of Clause 20, wherein the imaging chamber comprises one or more dyes that stain cells present in the biological sample.

[0223] Clause 23. The analysis cartridge of Clause 22, wherein the one or more dyes are spray dried into the imaging chamber.

[0224] Clause 24. The analysis cartridge of Clause 22, wherein the one or more dyes in the imaging chamber are in a coating that releases the one or more dyes.

[0225] Clause 25. The analysis cartridge of Clause 24, wherein the coating is a delayed- release coating.

[0226] Clause 26. The analysis cartridge of Clause 24 or 25, wherein the coating comprises the one or more dyes and one or both of ethyl cellulose and hydroxypropyl methyl cellulose (HPMC).

[0227] Clause 27. The analysis cartridge of any one of Clauses 21 to 26, wherein the one or more dyes comprise one or more of eosin, methylene blue, acridine orange, astrazon orange, and a dye to bind to nuclear DNA in the cells of the sample.

[0228] Clause 28. The analysis cartridge of any one of Clauses 20 to 27, wherein the sample loading port comprises a micro-mixer.

[0229] Clause 29. The analysis cartridge of any one of Clauses 1 to 28, wherein the chemical analysis module comprises a fluid distribution channel that distributes the non-cellular constituents of the biological sample into a plurality of detection chambers that detect a plurality of non-cellular constituents.

[0230] Clause 30. The analysis cartridge of Clause 29, further comprising a diluent blister that provides a diluent to produce a diluted sample for analysis of the non-cellular constituents.

[0231] Clause 31. The analysis cartridge of Clause 30, further comprising a diluent metering chamber that holds a metered amount of the diluent.

[0232] Clause 32. The analysis cartridge of any one of Clauses 29 to 31, further comprising a sample metering chamber for holding a metered amount of the non-cellular constituents.

[0233] Clause 33. The analysis cartridge of any one of Clauses 29 to 32, further comprising a diluent mixing chamber that mix the diluent with the non-cellular constituents.

[0234] Clause 34. The analysis cartridge of any one of Clauses 29 to 33, wherein the detection chambers comprise reagents that analyze specific constituents within the non-cellular constituents of the biological sample.

[0235] Clause 35. The analysis cartridge of any one of Clauses 1 to 34, wherein the chemical analysis module comprises a top panel and a bottom panel and a plurality of channels disposed between the top panel and the bottom panel, wherein the plurality of channels are configured for analyzing a plurality of non-cellular constituents.

[0236] Clause 36. The analysis cartridge of Clause 35, wherein the plurality of channels comprise reagents that analyze specific constituents within the non-cellular constituents of the biological sample.

[0237] Clause 37. A method for analyzing a biological sample for cells and non-cellular constituents, the method comprising introducing the biological sample into the analysis cartridge of any one of Clauses 1 to 36, and analyzing the biological sample.

[0238] Clause 38. The method of Clause 37, comprising introducing the biological sample into the sample inlet of a cellular analysis module and delivering the biological sample to the imaging chamber of the cellular analysis module.

[0239] Clause 39. The method of Clause 38, further comprising obtaining images of the cells in the sample delivered to the imaging chamber and analyzing the images to estimate the types and / or numbers of the cells present in the biological sample.

[0240] Clause 40. The method of any one of Clauses 37 to 39, comprising causing a portion of the biological sample introduced into the sample inlet of the cellular analysis module to transfer to the filtration module, causing the portion of the biological sample transferred to the filtration module to be filtered, and introducing the filtered biological sample into the sample inlet of the chemical analysis module.

[0241] Clause 41. The method of Clause 40, further comprising causing the sample to be loaded into a sample metering chamber.

[0242] Clause 42. The method of Clause 40 or 41, further comprising causing a diluent from a diluent blister to be loaded into a diluent metering chamber.

[0243] Clause 43. The method of Clause 42, further comprising mixing an appropriate amount of the diluent with an appropriate amount of the sample to produce a diluted sample in a diluent mixing chamber.

[0244] Clause 44. The method of Clause 43, further comprising distributing the diluted sample to a number of detection chambers via the sample distribution channel and thereby filling the plurality of detection chambers with the diluted sample.

[0245] Clause 45. The method of Clause 44, further comprising detecting and / or quantifying a plurality of non-cellular constituents of the sample by quantifying signals from the plurality of detection chambers.

[0246] Clause 46. A method for analyzing a biological sample for cells and non-cellular constituents, the method comprising:

[0247] introducing the biological sample into the sample inlet of a cellular analysis module,

[0248] transferring the biological sample to a sample loading port and delivering the biological sample into an imaging chamber of the cellular analysis module,

[0249] obtaining images of the cells in the loaded sample and analyzing the images to estimate the types and / or numbers of the cells present in the biological sample,

[0250] transferring a portion of the biological sample introduced into the sample inlet of the cellular analysis module to a filtration module,

[0251] filtering the portion of the biological sample transferred to the filtration module,

[0252] introducing the filtered biological sample into a sample inlet of a chemical analysis module,

[0253] loading the sample into a sample metering chamber,

[0254] loading a diluent from a diluent blister into a diluent metering chamber,

[0255] mixing in a diluent mixing chamber an appropriate amount of the diluent with an appropriate amount of the sample to produce a diluted sample,

[0256] distributing the diluted sample into a number of detection chambers via a sample distribution channel thereby filling a plurality of detection chambers with the diluted sample,

[0257] quantifying signals from the plurality of detection chambers and detecting and / or quantifying the plurality of non-cellular constituents.

[0258] Clause 47. An analyzer for analyzing a biological sample, the analyzer comprising a non-transitory storage media comprising instructions, which when executed by a computer, cause the analyzer to introduce the biological sample into the analysis cartridge of any one of Clauses 1 to 36 and analyze the biological sample.

[0259] Clause 48. The analyzer of Clause 47, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to introduce the biological sample into the sample inlet of the cellular analysis module and deliver the biological sample to the imaging chamber of the cellular analysis module.

[0260] Clause 49. The analyzer of Clause 48, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to obtain images of the cells in the sample delivered to the imaging chamber and analyze the images to estimate the types and / or numbers of the cells present in the biological sample.

[0261] Clause 50. The analyzer of any one of Clauses 47 to 49, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to transfer a portion of the biological sample introduced into the sample inlet of the cellular analysis module to the filtration module, cause the portion of the biological sample transferred to the filtration module to be filtered, and introduce the filtered biological sample into the sample inlet of the chemical analysis module.

[0262] Clause 51. The analyzer of Clause 50, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to cause the sample to be loaded into the sample metering chamber.

[0263] Clause 52. The analyzer of Clause 50 or 51, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to load a diluent from the diluent blister into the diluent metering chamber.

[0264] Clause 53. The analyzer of Clause 52, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to mix an appropriate amount of the diluent with an appropriate amount of the sample to produce a diluted sample in a diluent mixing chamber.

[0265] Clause 54. The analyzer of Clause 53, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to distribute the diluted sample to a number of detection chambers via the sample distribution channel and thereby filling the plurality of detection chambers with the diluted sample.

[0266] Clause 55. The analyzer of Clause 54, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to detect and / or quantify a plurality of non-cellular constituents of the sample by quantifying signals from the plurality of detection chambers.

[0267] Clause 56. An analyzer for analyzing a biological sample in the analysis cartridge of any one of Clauses 1 to 36, the analyzer comprising a non-transitory storage media comprising instructions, which when executed by a computer, cause the analyzer to:

[0268] introduce the biological sample into the sample inlet of a cellular analysis module,

[0269] transfer the biological sample to a sample loading port and delivering the biological sample into an imaging chamber of the cellular analysis module,

[0270] obtain images of the cells in the loaded sample and analyze the images to estimate the types and / or numbers of the cells present in the biological sample,

[0271] transfer a portion of the biological sample introduced into the sample inlet of the cellular analysis module to a filtration module,

[0272] filter the portion of the biological sample transferred to the filtration module,

[0273] introduce the filtered biological sample into a sample inlet of a chemical analysis module,

[0274] load the sample into a sample metering chamber,

[0275] load a diluent from a diluent blister into a diluent metering chamber,

[0276] mix in a diluent mixing chamber an appropriate amount of the diluent with an appropriate amount of the sample to produce a diluted sample,

[0277] distribute the diluted sample into a number of detection chambers via a sample distribution channel thereby filling a plurality of detection chambers with the diluted sample,

[0278] quantify signals from the plurality of detection chambers and detect and / or quantify the plurality of non-cellular constituents.

[0279] The preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

CLAIMSWE CLAIM:

1. An analysis cartridge for analyzing a biological sample, the analysis cartridge comprising: a cellular analysis module for analyzing cells of the biological sample, a chemical analysis module for analyzing non-cellular constituents of the biological sample, and a filtration module that filters the biological sample by transferring the biological sample from the cellular analysis module through a filter to the chemical analysis module thereby delivering the non-cellular constituents of the biological sample to the chemical analysis module.

2. The analysis cartridge of claim 1, wherein the filtration module comprises a first end fluidically connected to a sample inlet of the cellular analysis module and a second end fluidically connected to a sample inlet of the chemical analysis module, and a filter disposed between the first end and the second end.

3. The analysis cartridge of claim 1 or 2, wherein the cellular analysis module is stacked above the chemical analysis module.

4. The analysis cartridge of claim 3, wherein: the cellular analysis module is on a first substrate, the chemical analysis module is on a second substrate, the first substrate stacked above the second substrate, and the first end of the filtration module is fluidically connected to the sample inlet of the cellular analysis module and the second end of the filtration module is fluidically connected to the sample inlet of the chemical analysis module, and the filter is disposed below the cellular analysis module, and wherein the filtration module filters the biological sample as the biological sample transfers, under the force of gravity, from the sample inlet of the cellular analysis module through the filter to the sample inlet of the chemical analysis module.

5. The analysis cartridge of claim 1 or 2, wherein the chemical analysis module is stacked above the cellular analysis module.

6. The analysis cartridge of claim 5, wherein: the chemical analysis module is on a third substrate, the cellular analysis module is on a fourth substrate, the third substrate stacked above the fourth substrate, and the first end of the filtration module is fluidically connected to the sample inlet of the cellular analysis module and the second end of the filtration module is fluidically connected to the sample inlet of the chemical analysis module, and the filter is disposed above the cellular analysis module, and wherein the filtration module filters the biological sample as the biological sample transfers, under a force that moves the biological fluid against the force of gravity, from the sample inlet of the cellular analysis module through the filter to the sample inlet of the chemical analysis module.

7. The analysis cartridge of claim 6, wherein the filtration module further comprises a force generator that generates the force to move the biological sample against the force of gravity.

8. The analysis cartridge of claim 7, wherein the force generator is a pump.

9. The analysis cartridge of claim 8, wherein the pump is a vacuum pump, a peristaltic pump, or a diaphragm pump.

10. The analysis cartridge of claim 1 or 2, wherein the cellular analysis and the chemical analysis module are in substantially the same horizontal plane and are connected by the filtration module.

11. The analysis cartridge of claim 10, wherein the filtration module comprises a first end fluidically connected to a sample inlet of the cellular analysis module and a second end fluidically connected to a sample inlet of the chemical analysis module, and a filter disposed between the first end and the second end.

12. The analysis cartridge of claim 11, wherein the first end of the filtration module is fluidically connected to the sample inlet of the cellular analysis module and the second end of the filtration module is fluidically connected to the sample inlet of the chemical analysis module, and wherein the filtration module filters the biological sample as the biological sample transfers fromthe sample inlet of the cellular analysis module through the filter to the sample inlet of the chemical analysis module.

13. The analysis cartridge of claim 12, wherein the filtration module further comprises a force generator that generates the force to move the biological sample from the sample inlet of the cellular analysis module to the sample inlet of the chemical analysis module.

14. The analysis cartridge of claim 13, wherein the force generator is a pump.

15. The analysis cartridge of claim 14, wherein the pump is a vacuum pump, a peristaltic pump, or a diaphragm pump.

16. The analysis cartridge of any one of claims 1 to 15, wherein the fluidic connection between the sample inlet of the cellular analysis module and the filtration module comprises a diverter that diverts a portion of the biological sample introduced into the sample inlet of the cellular analysis module to the filtration module.

17. The analysis cartridge of any one of claims 1 to 16, wherein the filtration module comprises a membrane filter that filters cells from the non-cellular portion of the biological sample.

18. The analysis cartridge of claim 16, further comprising a hemoglobin detection chamber fluidically connected to the diverter.

19. The analysis cartridge of any one of claims 1 to 18, wherein the cellular analysis module comprises an imaging chamber comprising a top panel and a bottom panel separated by a suitable distance to produce a monolayer of cells between the panels.

20. The analysis cartridge of claim 19, wherein the imaging chamber comprises: a sample delivery edge where the gap between the top panel and the bottom panel is accessible to the biological sample delivered to the sample delivery edge, and a sample loading port delivers the biological sample to the sample delivery edge thereby allowing the biological sample to fill the imaging chamber.

21. The analysis cartridge of claim 20, wherein the sample loading port contains one or more dyes that stain cells present in the biological sample.

22. The analysis cartridge of claim 20, wherein the imaging chamber comprises one or more dyes that stain cells present in the biological sample.

23. The analysis cartridge of claim 22, wherein the one or more dyes are spray dried into the imaging chamber.

24. The analysis cartridge of claim 22, wherein the one or more dyes in the imaging chamber are in a coating that releases the one or more dyes.

25. The analysis cartridge of claim 24, wherein the coating is a delayed-release coating.

26. The analysis cartridge of claim 24 or 25, wherein the coating comprises the one or more dyes and one or both of ethyl cellulose and hydroxypropyl methyl cellulose (HPMC).

27. The analysis cartridge of any one of claims 21 to 26, wherein the one or more dyes comprise one or more of eosin, methylene blue, acridine orange, astrazon orange, and a dye to bind to nuclear DNA in the cells of the sample.

28. The analysis cartridge of any one of claims 20 to 27, wherein the sample loading port comprises a micro-mixer.

29. The analysis cartridge of any one of claims 1 to 28, wherein the chemical analysis module comprises a fluid distribution channel that distributes the non-cellular constituents of the biological sample into a plurality of detection chambers that detect a plurality of non-cellular constituents.

30. The analysis cartridge of claim 29, further comprising a diluent blister that provides a diluent to produce a diluted sample for analysis of the non-cellular constituents.31 . The analysis cartridge of claim 30, further comprising a diluent metering chamber that holds a metered amount of the diluent.

32. The analysis cartridge of any one of claims 29 to 31, further comprising a sample metering chamber for holding a metered amount of the non-cellular constituents.

33. The analysis cartridge of any one of claims 29 to 32, further comprising a diluent mixing chamber that mix the diluent with the non-cellular constituents.

34. The analysis cartridge of any one of claims 29 to 33, wherein the detection chambers comprise reagents that analyze specific constituents within the non-cellular constituents of the biological sample.

35. The analysis cartridge of any one of claims 1 to 34, wherein the chemical analysis module comprises a top panel and a bottom panel and a plurality of channels disposed between the top panel and the bottom panel, wherein the plurality of channels are configured for analyzing a plurality of non-cellular constituents.

36. The analysis cartridge of claim 35, wherein the plurality of channels comprise reagents that analyze specific constituents within the non-cellular constituents of the biological sample.

37. A method for analyzing a biological sample for cells and non-cellular constituents, the method comprising introducing the biological sample into the analysis cartridge of any one of claims 1 to 36, and analyzing the biological sample.

38. The method of claim 37, comprising introducing the biological sample into the sample inlet of a cellular analysis module and delivering the biological sample to the imaging chamber of the cellular analysis module.

39. The method of claim 38, further comprising obtaining images of the cells in the sample delivered to the imaging chamber and analyzing the images to estimate the types and / or numbers of the cells present in the biological sample.

40. The method of any one of claims 37 to 39, comprising causing a portion of the biological sample introduced into the sample inlet of the cellular analysis module to transfer tothe filtration module, causing the portion of the biological sample transferred to the filtration module to be filtered, and introducing the filtered biological sample into the sample inlet of the chemical analysis module.

41. The method of claim 40, further comprising causing the sample to be loaded into a sample metering chamber.

42. The method of claim 40 or 41, further comprising causing a diluent from a diluent blister to be loaded into a diluent metering chamber.

43. The method of claim 42, further comprising mixing an appropriate amount of the diluent with an appropriate amount of the sample to produce a diluted sample in a diluent mixing chamber.

44. The method of claim 43, further comprising distributing the diluted sample to a number of detection chambers via the sample distribution channel and thereby filling the plurality of detection chambers with the diluted sample.

45. The method of claim 44, further comprising detecting and / or quantifying a plurality of non-cellular constituents of the sample by quantifying signals from the plurality of detection chambers.

46. A method for analyzing a biological sample for cells and non-cellular constituents, the method comprising: introducing the biological sample into the sample inlet of a cellular analysis module, transferring the biological sample to a sample loading port and delivering the biological sample into an imaging chamber of the cellular analysis module, obtaining images of the cells in the loaded sample and analyzing the images to estimate the types and / or numbers of the cells present in the biological sample, transferring a portion of the biological sample introduced into the sample inlet of the cellular analysis module to a filtration module, filtering the portion of the biological sample transferred to the filtration module, introducing the filtered biological sample into a sample inlet of a chemical analysis module,loading the sample into a sample metering chamber, loading a diluent from a diluent blister into a diluent metering chamber, mixing in a diluent mixing chamber an appropriate amount of the diluent with an appropriate amount of the sample to produce a diluted sample, distributing the diluted sample into a number of detection chambers via a sample distribution channel thereby filling a plurality of detection chambers with the diluted sample, quantifying signals from the plurality of detection chambers and detecting and / or quantifying the plurality of non-cellular constituents.

47. An analyzer for analyzing a biological sample, the analyzer comprising a non- transitory storage media comprising instructions, which when executed by a computer, cause the analyzer to introduce the biological sample into the analysis cartridge of any one of claims 1 to 36 and analyze the biological sample.

48. The analyzer of claim 47, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to introduce the biological sample into the sample inlet of the cellular analysis module and deliver the biological sample to the imaging chamber of the cellular analysis module.

49. The analyzer of claim 48, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to obtain images of the cells in the sample delivered to the imaging chamber and analyze the images to estimate the types and / or numbers of the cells present in the biological sample.

50. The analyzer of any one of claims 47 to 49, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to transfer a portion of the biological sample introduced into the sample inlet of the cellular analysis module to the filtration module, cause the portion of the biological sample transferred to the filtration module to be filtered, and introduce the filtered biological sample into the sample inlet of the chemical analysis module.

51. The analyzer of claim 50, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to cause the sample to be loaded into the sample metering chamber.

52. The analyzer of claim 50 or 51, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to load a diluent from the diluent blister into the diluent metering chamber.

53. The analyzer of claim 52, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to mix an appropriate amount of the diluent with an appropriate amount of the sample to produce a diluted sample in a diluent mixing chamber.

54. The analyzer of claim 53, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to distribute the diluted sample to a number of detection chambers via the sample distribution channel and thereby filling the plurality of detection chambers with the diluted sample.

55. The analyzer of claim 54, wherein the non-transitory storage media further comprise instructions, which when executed by a computer, cause the analyzer to detect and / or quantify a plurality of non-cellular constituents of the sample by quantifying signals from the plurality of detection chambers.

56. An analyzer for analyzing a biological sample in the analysis cartridge of any one of claims 1 to 36, the analyzer comprising a non-transitory storage media comprising instructions, which when executed by a computer, cause the analyzer to: introduce the biological sample into the sample inlet of a cellular analysis module, transfer the biological sample to a sample loading port and delivering the biological sample into an imaging chamber of the cellular analysis module, obtain images of the cells in the loaded sample and analyze the images to estimate the types and / or numbers of the cells present in the biological sample, transfer a portion of the biological sample introduced into the sample inlet of the cellular analysis module to a filtration module, filter the portion of the biological sample transferred to the filtration module, introduce the filtered biological sample into a sample inlet of a chemical analysis module, load the sample into a sample metering chamber, load a diluent from a diluent blister into a diluent metering chamber,mix in a diluent mixing chamber an appropriate amount of the diluent with an appropriate amount of the sample to produce a diluted sample, distribute the diluted sample into a number of detection chambers via a sample distribution channel thereby filling a plurality of detection chambers with the diluted sample, quantify signals from the plurality of detection chambers and detect and / or quantify the plurality of non-cellular constituents.

Images