Apparatus and method for serial dilution of sample mixtures

Abstract

The present disclosure relates to a device and a method for continuously diluting a sample mixture, the device comprising a fluidic cartridge comprising: a first chamber configured to receive a sample and a first reagent to form a sample mixture (1); a metering chamber configured to meter a first portion of the sample mixture (1); a third chamber configured to receive a second portion of the sample mixture (1) and a third reagent to form a sample mixture (3); wherein the metering chamber is connected to the first chamber, and the third reagent is a dry or dehydrated reagent. Thus, the sample mixture can be continuously diluted by the device.

Description

[0001] This application is a divisional application of the patent application filed on February 26, 2019, with application number 201980015365.2 and entitled "Apparatus and Method for Sample Analysis Using Continuous Dilution". Technical Field

[0002] This disclosure generally relates to the biopharmaceutical industry, and specifically to an apparatus and method for continuously diluting sample mixtures. Background Technology

[0003] All publications cited herein are incorporated herein by reference in their entirety, just as each individual publication or patent application is specifically and individually indicated to be incorporated herein by reference. The following description includes information that may be used to understand this disclosure. This does not imply that any information provided herein is prior art or that any publication relating to this disclosure or any specifically or implicitly cited publication is prior art.

[0004] Sample analysis is typically a crucial step in in vitro diagnostics. Various types of samples can be analyzed, including but not limited to gaseous samples (e.g., breathed air), liquid samples such as bodily fluids (e.g., blood, lymph, sweat, tears, semen, saliva, and urine), and solid samples such as nucleic acid extracts or tumor biopsies. When the concentration of the analyte (e.g., cells, particles, biomolecules, and metabolites) in a sample is high, there is a significant possibility of introducing measurement errors during sample analysis. Therefore, it is necessary to dilute the sample to reduce measurement errors. A single-step dilution usually requires a large amount of reagent to dilute the sample for accurate analysis. In contrast, sequential dilution can reduce the amount of reagent required, thus making sample analysis easier and more practical.

[0005] Various types of sample analysis techniques can be used, including but not limited to cell counting, spectroscopic methods (e.g., mass spectrometry, optical spectrometry, and ion mobility spectrometry), and chemiluminescence methods. Cell counting is a technique used to measure cellular characteristics and is widely used in testing biological or medical samples. Cell counting can measure individual cells to obtain accurate characterization. However, when the cell concentration in a sample is high, there is a significant possibility that multiple cells may be characterized as a single cell, thus requiring sample dilution to reduce this possibility. In some cases, a single-step dilution will use a significant amount of diluent to dilute the high concentration of cells, while sequential dilution can reduce the amount of diluent required.

[0006] One example of cell counting analysis that benefits from serial dilution is the complete blood count (CBC) test. The CBC test measures the concentrations of white blood cells (WBC), red blood cells (RBC), and platelets (PLT) in a blood sample. In blood samples (such as human blood samples), the concentrations of blood cells and platelets are typically high (e.g., 4–6 million red blood cells and 0.15–0.5 million platelets per microliter of blood), often requiring serial dilution to dilute the blood sample for accurate measurements.

[0007] In addition, a complete blood count (CBC) can further measure parameters such as the sample's hemoglobin concentration and hematocrit. Sometimes, it may further classify white blood cells into different subtypes, such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils, and measure the concentration of these subtypes. Sometimes a CBC may further measure other parameters, such as hemoglobin, hematocrit, reticulocyte count, nucleated cell count, blood cell indices (e.g., mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and red blood cell distribution width), and platelet indices (e.g., mean platelet volume, plateletcrit, platelet distribution width, and large platelet cell percentage).

[0008] In the past, serial dilutions for sample analysis, such as cell counting analysis (e.g., complete blood count tests), were typically operated by technicians (e.g., by manual pipetting) or by automated machines (e.g., machines with built-in fluid systems). The fluid systems used for dilution in these automated machines are usually designed for continuous use rather than single-use and require frequent cleaning (e.g., after each sample measurement) and regular maintenance (e.g., bleaching biological residues). Therefore, these machines are mostly limited to use in central laboratories. For applications where convenience is paramount (e.g., point-of-care testing near patients), serial dilutions tend to be performed in single-use cartridges.

[0009] U.S. Patent Nos. 5,077,017 and 5,104,813 describe designs for cartridge devices for continuous dilution that rely on capillary forces to hold the sample's meniscus at the fluid confluence and on gravity to allow the diluent's meniscus to enter the sample's meniscus at the fluid confluence. Thus, the sum of capillary forces and gravity allows the sample and diluent to pass through the fluid confluence into the mixing chamber. The operation of this design is susceptible to air gaps between the sample's and diluent's meniscus at the fluid confluence, which can prevent direct contact between the two meniscus. These air gaps can be introduced from various sources, such as air bubbles in the sample (e.g., air bubbles in blood collected from a finger prick) or in the diluent (e.g., air bubbles generated during transport due to shaking) when the sample and diluent arrive at the fluid confluence.

[0010] U.S. Patent Nos. 9,440,233 and 9,808,802 describe cartridge designs for continuous dilution that rely on rotary valves to mix the sample and diluent together to form a mixture.

[0011] U.S. Patent Application No. 12 / 029,480 describes a cartridge design for continuous dilution that relies on the rotation and centrifugal force of the cartridge device to bring the sample and diluent together to form a mixture.

[0012] U.S. Patent Nos. 8,383,043, 8,518,328, 8,663,583, 8,741,235, and 8,980,635 describe cassette devices for cell counting analysis or complete blood cell count testing. However, they do not teach sequential dilution.

[0013] U.S. Patent Nos. 7,771,658 and 8,573,033 describe cassette devices for complete blood cell count testing that rely on rotary valves for operation. However, they do not teach continuous dilution.

[0014] U.S. Patent No. 9,625,357 describes a cassette device for a complete blood count test, but does not teach serial dilution.

[0015] U.S. Patent No. 5,627,041 describes a cassette device for metering samples for biological assays, but does not teach continuous dilution. Summary of the Invention

[0016] The following embodiments and aspects thereof will be described and illustrated in conjunction with apparatus, systems and methods, and are merely exemplary and illustrative and are not intended to limit the scope.

[0017] Various embodiments of this disclosure provide methods for analyzing samples. The method includes: mixing a sample with a first reagent to form a sample mixture (1); mixing a first portion of the sample mixture (1) with a second reagent to form a sample mixture (2); and measuring the sample mixture (1) or the sample mixture (2) or both to analyze cells, particles, or analytes, or combinations thereof. In various embodiments, the method further includes mixing a second portion of the sample mixture (1) with a third reagent to form a sample mixture (3), and measuring the sample mixture (3) to analyze cells, particles, or analytes, or combinations thereof. In various embodiments, the method further includes measuring a specified volume of the sample mixture (1) to obtain a first portion of the sample mixture (1). In various embodiments, the method further includes measuring a specified volume of the sample mixture (1) to obtain a second portion of the sample mixture (1).

[0018] Various embodiments of this disclosure provide apparatus for analyzing samples. In various embodiments, the apparatus includes a fluid cartridge. In various embodiments, the fluid cartridge includes: a first chamber configured to receive a sample and a first reagent to form a sample mixture (1); and a metering chamber connected to the first chamber and configured to meter a first portion of the sample mixture (1). In various embodiments, the fluid cartridge further includes a fluid structure configured to mix the first portion of the sample mixture (1) with a second reagent to form a sample mixture (2). In various embodiments, the fluid cartridge further includes a fluid structure configured to mix a second portion of the sample mixture (1) with a third reagent to form a sample mixture (3).

[0019] Various embodiments of this disclosure provide methods for analyzing samples. The method uses a fluid cartridge and a reader as described herein. The method includes: receiving a sample using a fluid cartridge as described herein; and performing sample analysis of the sample using a reader as described herein. In some embodiments, the fluid cartridge receives the sample before being placed into the reader. In other embodiments, the fluid cartridge receives the sample after being placed into the reader. Various embodiments of this disclosure provide methods for analyzing samples. The method includes: receiving a sample using a fluid cartridge; and placing the fluid cartridge into a reader for sample analysis of the sample. Various embodiments of this disclosure provide methods for analyzing samples. The method includes: placing a fluid cartridge into a reader; receiving a sample using the fluid cartridge; and performing sample analysis of the sample using the reader.

[0020] Various embodiments of this disclosure provide methods for analyzing samples. The method includes: using a fluid cartridge to receive a sample and a first reagent to form a sample mixture (1); using a metering chamber in the fluid cartridge to meter a first portion of the sample mixture (1); using the fluid cartridge to mix the first portion of the sample mixture (1) with a second reagent to form a sample mixture (2); and using a reader to measure a signal from the sample mixture (1) or the sample mixture (2) or both in the fluid cartridge. In various embodiments, the method further includes using the fluid cartridge to mix a second portion of the sample mixture (1) with a third reagent to form a sample mixture (3), and using the reader to measure a signal from the sample mixture (3).

[0021] Various embodiments of this disclosure provide apparatus and methods for serial dilution in various types of sample analysis, including, but not limited to, cell counting analysis (e.g., complete blood count tests). In various embodiments, this disclosure describes dilution methods and fluidic diagrams for implementing serial dilution in sample analysis (e.g., cell counting analysis and complete blood count tests). In various embodiments, this disclosure describes apparatus for fluid cartridges to implement serial dilution for sample analysis (e.g., cell counting analysis and complete blood count tests). In various embodiments, this disclosure describes reagent storage units for fluid cartridge apparatuses to extend their shelf life. In some embodiments, the fluid cartridge is received into a reader for measurement operations and result reading. In addition to cell counting analysis and complete blood count tests, the serial dilution methods, fluid cartridge apparatuses, and reagent storage units can be used for other measurements of samples. Examples of other measurements include, but are not limited to, hemoglobin, hematocrit, reticulocyte count, nucleated red blood cell count, erythrocyte indices (e.g., mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and erythrocyte distribution width), and platelet indices (e.g., mean platelet volume, plateletcrit, platelet distribution width, and platelet-large cell ratio). Furthermore, the apparatus and methods described herein can also be used to analyze various types of samples, including but not limited to bodily fluids (e.g., blood, sweat, tears, and urine), other fluid samples (e.g., cell suspensions in buffer or particle suspensions in buffer), and other types of samples (e.g., nucleic acid extracts or tumor biopsies). In various embodiments, the apparatus and methods described in this disclosure can also be used to perform metrology of sample mixtures, serial dilution of sample mixtures, and various analyses of samples. Attached Figure Description

[0022] Exemplary embodiments are illustrated in the accompanying drawings. The embodiments and drawings disclosed herein should be considered illustrative rather than restrictive.

[0023] Figure 1A This is a block diagram illustrating the order of dilution in various sample analysis methods, including but not limited to cell counting analysis.

[0024] Figure 1B This is a block diagram used to illustrate the order of dilution in cell counting analysis and other measurements.

[0025] Figure 2 This illustrates a fluid diagram showing the implementation of serial dilutions in various sample analysis methods (e.g., cell counting analysis and other measurements).

[0026] Figure 3 An example of a flow chamber used for cell counting analysis is shown.

[0027] Figure 4 An example of a sensing area used for transmittance measurement is shown.

[0028] Figures 5A-5C An example of a fluidic cartridge device is shown for analysis of samples with continuous dilution (e.g., cell counting analysis).

[0029] Figure 5D-5F An example of a fluid structure configured to meter a portion of a sample mixture is shown, which can be used for continuous dilution and sample analysis.

[0030] Figure 5G-5K An example of a fluid structure configured for continuous dilution of a sample mixture that can be used for sample analysis is shown.

[0031] Figures 6A-6F An example of a passive valve is shown in a fluid cartridge used for sample analysis with continuous dilution (e.g., cell counting analysis).

[0032] Figures 7A-7D An example of a reagent storage unit is shown in a fluid cartridge used for sample analysis with continuous dilution (e.g., cell counting analysis).

[0033] Figure 8A The fluid cartridge device and the reader that receives the fluid cartridge device are shown.

[0034] Figure 8B The functional modules in the reader are shown.

[0035] Figure 8C This shows the orientation of the fluid cartridge device after it is received by the reader.

[0036] Figures 9A-9B An example of a whole blood cell count test result obtained using a serial dilution method in a fluid cartridge device is shown. Detailed Implementation

[0037] All references cited herein are incorporated herein by reference in their entirety, as if fully set forth herein. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Tabelling, *Introduction to Microfluidics* (2nd edition), Oxford University Press (2010); Hguyen et al., *Fundamentals and Applications of Microfluidics 2nd ed.*, Artech House Incorporated (2006); Berg et al., *Microfluidics for Medical Applications*, Royal Society of Chemistry (2014); Gomez et al., *Biological Applications of Microfluidics 1st ed.*, Wiley-Interscience (2008); and Colin et al., *Microfluidics 1st ed.*, Wiley-ISTE (2010) provide general guidance to those skilled in the art on many of the terms used in this application.

[0038] Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described herein can be used in practicing this disclosure. Further features and advantages of this disclosure will become apparent from the following detailed description taken in conjunction with the accompanying drawings, which illustrate various features of embodiments of this disclosure by way of example. In fact, this disclosure is by no means limited to the methods and materials described. For convenience, certain terms used herein in the specification, examples, and appended claims are collected.

[0039] Unless otherwise stated or implied by the context, the following terms and phrases include the meanings provided below. Unless otherwise expressly stated or obvious from the context, the following terms and phrases do not exclude the meaning that a term or phrase has acquired in the art to which it pertains. 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 to which this disclosure pertains. It should be understood that this disclosure is not limited to the specific methods, procedures, and reagents described herein, and therefore may vary. The definitions and terms used herein are for the purpose of describing particular embodiments and not for limiting the claims.

[0040] As used herein, the terms “comprising” or “comprises” are used to refer to compositions, methods, and their respective components, which may be used in the embodiments but include unspecified elements, whether useful or not. Those skilled in the art will understand that, in general, the terms used herein are intended to be “open-ended” (e.g., the term “including” should be interpreted as “including but not limited to”, the term “having” should be interpreted as “having at least”, the term “includes” should be interpreted as “including but not limited to”, etc.).

[0041] Unless otherwise stated, the terms “a”, “an”, and “the”, and similar references used in the context of describing particular embodiments of this application (particularly in the context of the claims) may be interpreted as covering both singular and plural. The description of numerical ranges herein is intended only as a shorthand method of individually referring to each individual value falling within that range. Each individual value is incorporated into this specification as if it were individually referenced herein, unless otherwise stated herein. All methods described herein may be performed in any suitable order unless otherwise stated herein or clearly contradicted by the context. The use of any and all examples or exemplary language (e.g., “such as”) provided herein with respect to certain embodiments is intended only to better illustrate the application and does not constitute a limitation on the scope of the claimed application. The abbreviation “e.g.” is derived from the Latin *exempli gratia* and is used herein to indicate a non-limiting example. Therefore, the abbreviation “e.g.” is synonymous with the term “for example.” No language in the specification should be construed as indicating any unclaimed element necessary for practicing this application.

[0042] Patent applications with application numbers PCT / US17 / 59965, 15 / 803,133, PCT / US17 / 62765, 15 / 819,416, 62 / 504,866, PCT / US18 / 31893, 62 / 575,918, and PCT / US18 / 56725 are all incorporated herein by reference as if fully explained.

[0043] Various embodiments of this disclosure provide methods for analyzing samples. The method includes: mixing a sample with a first reagent to form a sample mixture 1; mixing a first portion of the sample mixture 1 with a second reagent to form a sample mixture 2; and measuring the sample mixture 1, sample mixture 2, or both to analyze cells, particles, or analytes, or combinations thereof. In various embodiments, the method further includes mixing a second portion of the sample mixture 1 with a third reagent to form a sample mixture 3, and measuring the sample mixture 3 to analyze cells, particles, or analytes, or combinations thereof.

[0044] In various embodiments, a first chamber in the fluid cartridge is used to mix a sample with a first reagent to form sample mixture 1. In various embodiments, a fluid structure in the fluid cartridge is used to mix a first portion of sample mixture 1 with a second reagent to form sample mixture 2. In various embodiments, a fluid structure in the fluid cartridge is used to mix a second portion of sample mixture 1 with a third reagent to form sample mixture 3.

[0045] In various embodiments, the method further includes metering a specified volume of sample mixture 1 to obtain a first portion of sample mixture 1. In various embodiments, the method further includes metering a specified volume of sample mixture 1 to obtain a second portion of sample mixture 1. In various embodiments, a metering chamber and a drive mechanism are used to meter the first and / or second portions of sample mixture 1.

[0046] In various embodiments, the method further includes forming a sample stream from the sample mixture and measuring cells, particles, or analytes, or combinations thereof, in the sample stream. In various embodiments, a flow chamber is used to form the sample stream. In various embodiments, the sample mixture is any sample mixture in a fluidized bed. In some embodiments, the sample mixture is sample mixture 1. In some embodiments, the sample mixture is sample mixture 2. In some embodiments, the sample mixture is sample mixture 3. In various embodiments, sample mixture 2 is measured in the flow chamber before sample mixture 3 is measured in the flow chamber.

[0047] In various embodiments, the method further includes using a reader to measure cells, particles, or analytes, or combinations thereof, in the sample stream. In various embodiments, cells measured by the reader include white blood cells (WBCs), red blood cells (RBCs), or platelets (PLTs), or combinations thereof. In various embodiments, particles measured by the reader include lipid particles, beads, fluorescent beads, or magnetic beads, or combinations thereof. In various embodiments, analytes measured by the reader include hemoglobin, proteins, or hormones, or combinations thereof.

[0048] In various embodiments, sample mixture 2 is measured to analyze cells, red blood cells, platelets, particles, or analytes, or combinations thereof. In various embodiments, sample mixture 3 is measured to analyze cells, white blood cells, hemoglobin, particles, or analytes, or combinations thereof.

[0049] Various embodiments of this disclosure provide an apparatus for analyzing samples. In various embodiments, the apparatus includes a fluid cartridge. In various embodiments, the fluid cartridge includes: a first chamber configured to receive a sample and a first reagent to form a sample mixture 1; and a metering chamber connected to the first chamber and configured to meter a first portion of the sample mixture 1. In some embodiments, the first reagent is a liquid reagent. In other embodiments, the first reagent is a dried or dehydrated reagent. In various embodiments, the molar osmotic pressure concentration of the first reagent is approximately 140 mOsm / L to 160 mOsm / L, 160 mOsm / L to 180 mOsm / L, 180 mOsm / L to 200 mOsm / L, 200 mOsm / L to 220 mOsm / L, 220 mOsm / L to 240 mOsm / L, and 240 mOsm / L to 260 mOsm / L. / L, 260mOsm / L to 280mOsm / L, 280mOsm / L to 300mOsm / L, 300mOsm / L to 320mOsm / L, 320mOsm / L to 340mOsm / L, 340mOsm / L to 360mOsm / L, 360mOsm / L to 380mOsm / L or 380mOsm / L to 400mOsm / L.

[0050] In various embodiments, the fluid cartridge further includes a fluid structure configured to mix a first portion of sample mixture 1 with a second reagent to form sample mixture 2. In various embodiments, the fluid structure includes a second chamber connected to a metering chamber. In various embodiments, the second chamber is configured to receive a second reagent prior to mixing the second reagent with the first portion of sample mixture 1. In various embodiments, the fluid structure includes a second chamber connected to the metering chamber and configured to receive the second reagent prior to mixing the second reagent with the first portion of sample mixture 1. In various embodiments, the connection between the metering chamber and the second chamber includes a valve, a passive valve, or an active valve. In various embodiments, the fluid structure includes a second chamber connected to a first chamber. In various embodiments, the fluid cartridge further includes a fluid structure configured to mix a second portion of sample mixture 1 with a third reagent to form sample mixture 3.

[0051] In various embodiments, the metering chamber includes a surface, and at least a portion of the surface is hydrophilic. In various embodiments, the metering chamber is connected to a drive mechanism configured to drive the sample mixture 1 to contact the hydrophilic surface in the metering chamber. In various embodiments, the metering chamber includes a capillary valve. In various embodiments, the metering chamber is connected to a vent. In various embodiments, the metering chamber is connected to a chamber having a vent. In various embodiments, the connection between the metering chamber and the chamber having the vent includes a valve. In some embodiments, the valve is a capillary valve. In some embodiments, the valve is a passive valve, an active valve, or a combination thereof.

[0052] In various embodiments, the device further includes a reader configured to receive the fluid cartridge and perform sample analysis. In various embodiments, the reader is configured to receive the fluid cartridge by gravity, pulling the fluid within the chamber away from the chamber vent. In various embodiments, the reader is configured to receive the fluid cartridge by gravity, pulling the fluid within the chamber away from the chamber vent and towards the bottom of the chamber.

[0053] In various embodiments, the reader includes a drive mechanism. In various embodiments, the drive mechanism is pneumatic. In various embodiments, the drive mechanism is connected to a metering chamber. In various embodiments, the drive mechanism is configured to drive the sample mixture 1 to contact a hydrophilic surface in the metering chamber.

[0054] In various embodiments, the reader is configured to apply a drive mechanism to the fluid cartridge to measure a first portion of the sample mixture 1 in the metering chamber. In various embodiments, the drive mechanism is activated to drive the sample mixture 1 into contact with a hydrophilic surface in the metering chamber. In various embodiments, the drive mechanism is deactivated after the sample mixture 1 has contacted the hydrophilic surface in the metering chamber. In various embodiments, the drive mechanism is deactivated after the sample mixture 1 has contacted the hydrophilic surface in the metering chamber and before the sample mixture 1 reaches the capillary valve in the metering chamber.

[0055] In various embodiments, the fluid cartridge further includes a reagent storage unit comprising a storage chamber configured to contain fluid and a valve with a breakable seal. In various embodiments, the fluid cartridge further includes two reagent storage units, each comprising a storage chamber configured to contain fluid and a valve with a breakable seal. In various embodiments, after the fluid cartridge is placed in the reader, two valves in the two reagent storage units open simultaneously. In various embodiments, the storage chamber described herein is configured to contain a first reagent and / or a second reagent. In various embodiments, the fluid contained in the storage chamber as described herein is the first reagent and / or the second reagent.

[0056] In various embodiments, the fluid cartridge further includes a flow chamber configured to form a sample stream from the sample mixture in the fluid cartridge. In various embodiments, the sample mixture is any sample mixture in the fluid cartridge. In some embodiments, the sample mixture is sample mixture 1. In some embodiments, the sample mixture is sample mixture 2. In some embodiments, the sample mixture is sample mixture 3.

[0057] Various embodiments of this disclosure provide methods for analyzing samples. The method uses a fluid cartridge and a reader as described herein. The method includes: receiving a sample using the fluid cartridge as described herein; and performing sample analysis of the sample using the reader as described herein. In some embodiments, the fluid cartridge receives the sample before being placed into the reader. In other embodiments, the fluid cartridge receives the sample after being placed into the reader.

[0058] In various embodiments, the method includes: receiving a sample using a fluid cartridge; and placing the fluid cartridge into a reader for sample analysis of the sample. In various embodiments, the fluid cartridge includes: a first chamber configured to receive a sample and a first reagent to form a sample mixture 1; and a metering chamber connected to the first chamber and configured to meter a first portion of the sample mixture 1. In some embodiments, the first reagent is a liquid reagent. In other embodiments, the first reagent is a dried or dehydrated reagent.

[0059] In various embodiments, the method includes: placing a fluid cartridge into a reader; using the fluid cartridge to receive a sample; and using the reader to perform sample analysis on the sample. In various embodiments, the fluid cartridge includes: a first chamber configured to receive a sample and a first reagent to form a sample mixture 1; and a metering chamber connected to the first chamber and configured to meter a first portion of the sample mixture 1. In some embodiments, the first reagent is a liquid reagent. In other embodiments, the first reagent is a dried or dehydrated reagent.

[0060] In various embodiments, the molar osmotic concentration of the first reagent is approximately 140 mOsm / L to 160 mOsm / L, 160 mOsm / L to 180 mOsm / L, 180 mOsm / L to 200 mOsm / L, 200 mOsm / L to 220 mOsm / L, 220 mOsm / L to 240 mOsm / L, and 240 mOsm / L to 260 mOsm / L. L, 260mOsm / L to 280mOsm / L, 280mOsm / L to 300mOsm / L, 300mOsm / L to 320mOsm / L, 320mOsm / L to 340mOsm / L, 340mOsm / L to 360mOsm / L, 360mOsm / L to 380mOsm / L, or 380mOsm / L to 400mOsm / L.

[0061] In various embodiments, the metering chamber includes a surface, and at least a portion of the surface is hydrophilic. In various embodiments, the metering chamber is connected to a drive mechanism configured to drive the sample mixture 1 to contact the hydrophilic surface in the metering chamber. In various embodiments, the metering chamber includes a capillary valve. In various embodiments, the metering chamber is connected to a vent. In various embodiments, the metering chamber is connected to a chamber having a vent. In various embodiments, the connection between the metering chamber and the chamber having the vent includes a valve. In some embodiments, the valve is a capillary valve. In some embodiments, the valve is a passive valve, an active valve, or a combination thereof.

[0062] In various embodiments, the reader applies a drive mechanism to the fluid cartridge to meter a first portion of sample mixture 1 in the metering chamber. In various embodiments, the drive mechanism is pneumatic. In various embodiments, the drive mechanism is activated to drive sample mixture 1 into contact with a hydrophilic surface in the metering chamber. In various embodiments, the drive mechanism is deactivated after sample mixture 1 has contacted the hydrophilic surface in the metering chamber. In various embodiments, the drive mechanism is deactivated after sample mixture 1 has contacted the hydrophilic surface in the metering chamber and before sample mixture 1 reaches the capillary valve in the metering chamber.

[0063] In various embodiments, the fluid cartridge is placed in the reader by gravity, pulling the fluid within its chamber away from the chamber vent. In various embodiments, the fluid cartridge is placed in the reader by gravity, pulling the fluid within its chamber away from the chamber vent and towards the bottom of the chamber.

[0064] In various embodiments, after the first portion of the sample mixture 1 is metered, the sample mixture 1 is removed from the first chamber.

[0065] In various embodiments, the fluid cartridge further includes a fluid structure configured to mix a first portion of sample mixture 1 with a second reagent to form sample mixture 2. In various embodiments, the fluid structure includes a second chamber connected to the metering chamber. In various embodiments, the second chamber is configured to receive the second reagent prior to mixing the second reagent with the first portion of sample mixture 1. In various embodiments, the fluid structure includes a second chamber connected to the metering chamber and configured to receive the second reagent prior to mixing the second reagent with the first portion of sample mixture 1. In various embodiments, the connection between the metering chamber and the second chamber includes a valve, a passive valve, or an active valve. In various embodiments, the fluid structure includes a second chamber connected to the first chamber.

[0066] In various embodiments, the fluid cartridge is configured to mix a second portion of sample mixture 1 with a third reagent to form sample mixture 3. In various embodiments, the third reagent is a dried or dehydrated reagent.

[0067] In various embodiments, the fluid cartridge further includes a flow chamber configured to form a sample stream from the sample mixture in the fluid cartridge. In various embodiments, the fluid cartridge further includes a flow chamber configured to form a sample stream from the sample mixture in the fluid cartridge, and a reader measures cells, particles, or analytes, or combinations thereof, in the sample stream. In various embodiments, the sample mixture is any sample mixture in the fluid cartridge. In some embodiments, the sample mixture is sample mixture 1. In some embodiments, the sample mixture is sample mixture 2. In some embodiments, the sample mixture is sample mixture 3. In some embodiments, sample mixture 2 is measured in the flow chamber before sample mixture 3 is measured in the flow chamber.

[0068] In various embodiments, the cells measured by the reader include white blood cells (WBC), red blood cells (RBC), or platelets (PLT), or combinations thereof. In various embodiments, the particles measured by the reader include lipid particles, beads, fluorescent beads, or magnetic beads, or combinations thereof. In various embodiments, the analytes measured by the reader include hemoglobin, proteins, or hormones, or combinations thereof.

[0069] In various embodiments, sample mixture 2 is measured to analyze cells, red blood cells, platelets, particles, or analytes, or combinations thereof. In various embodiments, sample mixture 3 is measured to analyze cells, white blood cells, hemoglobin, particles, or analytes, or combinations thereof.

[0070] In various embodiments, the fluid cartridge further includes a reagent storage unit comprising a storage chamber configured to contain fluid and a valve with a breakable seal. In various embodiments, the fluid cartridge further includes two reagent storage units, each comprising a storage chamber configured to contain fluid and a valve with a breakable seal. In various embodiments, after the fluid cartridge is placed into the reader, two valves in the two reagent storage units open simultaneously. In various embodiments, the storage chamber described herein is configured to contain a first reagent and / or a second reagent. In various embodiments, the fluid contained in the storage chamber as described herein is the first reagent and / or the second reagent.

[0071] Various embodiments of this disclosure provide methods for analyzing samples. The method includes: using a fluid cartridge to receive a sample and a first reagent to form a sample mixture 1; using a metering chamber in the fluid cartridge to meter a first portion of the sample mixture 1; using the fluid cartridge to mix the first portion of the sample mixture 1 with a second reagent to form a sample mixture 2; and using a reader to measure signals from the sample mixture 1 or sample mixture 2, or both, in the fluid cartridge.

[0072] In various embodiments, the method further includes using a fluid cartridge to mix a second portion of sample mixture 1 with a third reagent to form sample mixture 3. In various embodiments, the third reagent includes a red blood cell lysis compound. In various embodiments, the method further includes using a reader to measure the signal from sample mixture 3.

[0073] In some embodiments, the reader measures sample mixture 2 to analyze cells, red blood cells, platelets, particles, or analytes, or combinations thereof. In some embodiments, the reader measures sample mixture 3 to analyze cells, white blood cells, hemoglobin, particles, or analytes, or combinations thereof. In various embodiments, the method further includes measuring sample mixture 2 in the flow chamber of the fluid cartridge before measuring sample mixture 3 in the flow chamber of the fluid cartridge.

[0074] In various embodiments, the reader applies a drive mechanism to the metering chamber to meter a first portion of sample mixture 1. In various embodiments, the drive mechanism is pneumatic. In various embodiments, the drive mechanism is activated to drive sample mixture 1 to contact a hydrophilic surface in the metering chamber. In various embodiments, the drive mechanism is stopped after sample mixture 1 contacts the hydrophilic surface in the metering chamber. In various embodiments, the drive mechanism is stopped after sample mixture 1 contacts the hydrophilic surface in the metering chamber and before sample mixture 1 reaches the capillary valve in the metering chamber.

[0075] In various embodiments, the fluid cartridge is placed within the reader. In various embodiments, the fluid cartridge is positioned within the reader by gravity, pulling the fluid within its chamber away from the chamber vent. In various embodiments, the fluid cartridge is positioned within the reader by gravity, pulling the fluid within its chamber away from the chamber vent and towards the bottom of the chamber.

[0076] In various embodiments, the molar osmotic concentration of the first reagent is approximately 140 mOsm / L to 160 mOsm / L, 160 mOsm / L to 180 mOsm / L, 180 mOsm / L to 200 mOsm / L, 200 mOsm / L to 220 mOsm / L, 220 mOsm / L to 240 mOsm / L, and 240 mOsm / L to 260 mOsm / L. 260 mOsm / L to 280 mOsm / L, 280 mOsm / L to 300 mOsm / L, 300 mOsm / L to 320 mOsm / L, 320 mOsm / L to 340 mOsm / L, 340 mOsm / L to 360 mOsm / L, 360 mOsm / L to 380 mOsm / L, or 380 mOsm / L to 400 mOsm / L.

[0077] In various embodiments, a red blood cell lysis compound is introduced into the chamber, and then a first portion of sample mixture 1 and a second reagent are introduced into the chamber to form sample mixture 2.

[0078] In various embodiments, the method further includes using a flow chamber in a fluidized bed to form a sample stream from a sample mixture in the fluidized bed, and using a reader to measure cells, particles, or analytes, or combinations thereof, in the sample stream. In various embodiments, the sample mixture is any sample mixture in the fluidized bed. In some embodiments, the sample mixture is sample mixture 1. In some embodiments, the sample mixture is sample mixture 2. In some embodiments, the sample mixture is sample mixture 3. In various embodiments, sample mixture 2 is measured in the flow chamber before sample mixture 3 is measured in the flow chamber. In various embodiments, the cells measured by the reader include white blood cells (WBC), red blood cells (RBC), or platelets (PLT), or combinations thereof. In various embodiments, the particles measured by the reader include lipid particles, beads, fluorescent beads, or magnetic beads, or combinations thereof. In various embodiments, the analytes measured by the reader include hemoglobin, proteins, or hormones, or combinations thereof.

[0079] In various embodiments, the method further includes using a reader to measure cells, red blood cells, platelets, particles, or analytes or combinations thereof in a sample stream formed from sample mixture 2. In various embodiments, the method further includes using a reader to measure cells, white blood cells, hemoglobin, particles, or analytes or combinations thereof in a sample stream formed from sample mixture 3.

[0080] In various embodiments, the method further includes using a flow chamber in a fluid cartridge to form a sample stream from a sample mixture 2 in the fluid cartridge, and using a reader to measure cells, red blood cells, platelets, particles, or analytes or combinations thereof in the sample stream formed from the sample mixture 2. In various embodiments, the method further includes using a flow chamber in a fluid cartridge to form a sample stream from a sample mixture 3 in the fluid cartridge, and using a reader to measure cells, white blood cells, hemoglobin, particles, or analytes or combinations thereof in the sample stream formed from the sample mixture 3.

[0081] In various embodiments, this disclosure provides methods for sample analysis (e.g., cell counting analysis) using a fluidic cartridge, the fluidic cartridge including a serial dilution mechanism or structure. In various embodiments, the methods described herein include: mixing a sample with a first reagent to form a sample mixture 1; metering a first portion of sample mixture 1 and mixing the first portion of sample mixture 1 with a second reagent to form a sample mixture 2; mixing a second portion of sample mixture 1 with a third reagent to form a sample mixture 3; using a flow chamber to form a sample stream from any sample mixture; and measuring a signal from the sample stream to detect cells. In various embodiments, the methods described herein further include using a metering chamber and a drive mechanism to meter a specified volume of sample mixture 1 to form sample mixture 2.

[0082] In various embodiments, the methods described herein use a serial dilution mechanism or structure to prepare sample mixtures. In various embodiments, the serial dilution mechanism or structure described herein mixes a sample (e.g., a blood sample) with a first reagent to form a first-dilution sample mixture 1. In various embodiments, the serial dilution mechanism or structure described herein measures a first portion of sample mixture 1 and mixes it with a second reagent to form a second-dilution sample mixture 2. In some embodiments, the serial dilution mechanism or structure described herein measures a second portion of sample mixture 1 and mixes it with a third reagent to form sample mixture 3. In other embodiments, the serial dilution mechanism or structure described herein mixes all remaining portions of sample mixture 1 (after removing the first-measured portion) with a third reagent to form sample mixture 3.

[0083] In some embodiments, the third reagent is a dried or dehydrated reagent. In other embodiments, the third reagent is a liquid reagent. In some embodiments, the third reagent is a liquid reagent, and after the first portion of sample mixture 1 is removed, the second portion of sample mixture 1 is the entire remaining volume of sample mixture 1.

[0084] In various embodiments, the continuous dilution mechanism or structure described herein uses a metering chamber and a drive mechanism to meter a specified volume of sample mixture 1 to form sample mixture 2. In some embodiments, the drive mechanism includes a hydrophilic surface and additional drive mechanisms (e.g., a pneumatic drive mechanism).

[0085] In various embodiments, the methods described herein use a flow chamber to measure signals from sample mixtures to detect cells. In some embodiments, the flow chamber measures both sample mixture 2 and sample mixture 3. In various embodiments, the methods described herein further include using the flow chamber to form two separate sample streams from sample mixture 2 and sample mixture 3, and measuring signals from both sample streams to detect cells. In some embodiments, the measurement order is: first sample mixture 2, then sample mixture 3. In some embodiments, the methods described herein further include introducing at least one gas gap between the measured sample mixtures (e.g., sample mixture 2 and sample mixture 3).

[0086] In various embodiments, the first and / or second reagents include a diluent, and the sample mixture 2 is measured to analyze red blood cells and / or platelets. In various embodiments, the third reagent includes a red blood cell lysis compound, and the sample mixture 3 is measured to analyze white blood cells or hemoglobin or both.

[0087] In various embodiments, the methods described herein use a fluid cartridge. In various embodiments, the fluid cartridge includes a continuous dilution mechanism or structure as described herein. In various embodiments, the fluid cartridge also includes a reagent storage unit.

[0088] In various embodiments, the reagent storage unit described herein stores a specified volume of reagent. In various embodiments, the reagent storage unit includes a storage chamber configured to contain a fluid reagent and a valve with a breakable seal. In some embodiments, the reagent storage unit further includes a vent with a breakable seal. In various embodiments, the reagent storage unit is connected to or includes a pneumatically driven mechanism. According to this disclosure, the reagent storage unit and / or a portion thereof includes a water vapor barrier material. In various embodiments, at least a portion of the storage chamber includes a water vapor barrier material. In various embodiments, the water vapor barrier material has a density of approximately 0.0001 g·mm / (m²). 2 ·day) to 0.01g·mm / (m 2 ·day), 0.01g·mm / (m 2 ·day) to 0.1g·mm / (m 2 ·day), 0.1g·mm / (m 2 ·day) to 1g·mm / (m 2 ·day) or 1g·mm / (m2 ·day) to 5g·mm / (m 2 Water vapor permeability (day) at 23°C and 85% relative humidity.

[0089] In various embodiments, the fluid cartridge described herein includes at least two reagent storage units. In some embodiments, at least one reagent storage unit is used for a first reagent, and at least another reagent storage unit is used for a second reagent. In various embodiments, each of the at least two reagent storage units includes a storage chamber configured to contain a fluid reagent and a valve with a breakable connection. In various embodiments, when the fluid cartridge is received into the reader, the valves in the at least two reagent storage units open simultaneously.

[0090] In various embodiments, the fluid cartridge described herein also includes a chamber, a valve, a fluid passage or a filter, or a combination thereof.

[0091] In various embodiments, the fluid cartridge described herein also includes regions for additional measurements (e.g., hemoglobin and hematocrit measurements). In various embodiments, the fluid cartridge described herein also includes an optical window for transmittance measurements. In some embodiments, transmittance measurements are used to analyze hemoglobin and / or hematocrit.

[0092] In various embodiments, the methods described herein utilize a reader that receives a fluid cartridge and performs cell measurements and / or analysis. In various embodiments, the methods described herein also include placing a fluid cartridge into the reader for cell measurements and / or analysis.

[0093] In various embodiments, the reader also includes a drive mechanism and / or a gate to block ambient light.

[0094] In various embodiments, the fluid cartridge received by the reader is positioned such that gravity pulls the fluid within the chamber away from the chamber vent and toward the bottom of the chamber. In various embodiments, the chamber is one or more of a first chamber, a second chamber, a third chamber, and a storage chamber. In some embodiments, the chamber is configured to store a fluid reagent. In this orientation, a gas gap is formed between the vent and the fluid reagent. Simultaneously, a channel accesses the chamber below the upper surface of the fluid reagent.

[0095] In various embodiments, the fluid cartridge received by the reader is positioned such that gravity pulls the fluid within the chamber away from the chamber vent and toward the bottom of the chamber. In various embodiments, the chamber is one or more of a first chamber, a second chamber, a third chamber, and a storage chamber. In some embodiments, the chamber is configured to form a diluted sample mixture. In this orientation, an air gap is formed between the vent and the diluted sample mixture. Simultaneously, a channel enters the chamber below the top surface of the diluted sample mixture. In some embodiments, the diluted sample mixture is transferred from the chamber to a flow chamber via the channel to form a sample flow for cell counting analysis.

[0096] In various embodiments, this disclosure provides a continuous dilution mechanism or structure. In various embodiments, the continuous dilution mechanism or structure is part of a fluid cartridge. In various embodiments, this disclosure provides an apparatus including a fluid cartridge that incorporates a continuous dilution mechanism or structure.

[0097] In various embodiments, the continuous dilution mechanism or structure described herein includes: a first chamber configured to mix a sample (e.g., a blood sample) with a first reagent to form a sample mixture 1; and a metering chamber connected to the first chamber and configured to meter a specified volume of the sample mixture 1. In various embodiments, the metering chamber has a hydrophilic surface and is connected to a drive mechanism configured to drive the sample mixture 1 to contact the hydrophilic surface. In some embodiments, the drive mechanism is a pneumatic drive mechanism. In various embodiments, the continuous dilution mechanism or structure further includes a second chamber connected to the metering chamber and configured to mix a metered portion of the sample mixture 1 with a second reagent to form a sample mixture 2.

[0098] In various embodiments, the serial dilution mechanism or structure described herein further includes a third chamber connected to the first chamber and configured for mixing a portion of sample mixture 1 with a third reagent to form sample mixture 3. In some embodiments, the serial dilution mechanism or structure further includes another metering chamber configured for metering a portion of sample mixture 1 for forming sample mixture 3. In some embodiments, the serial dilution mechanism or structure is configured for mixing a metered volume of sample mixture 1 with a third reagent to form sample mixture 3. In other embodiments, the serial dilution mechanism or structure is configured for mixing all remaining volumes of sample mixture 1 (after the first metered portion of sample mixture 1 has been removed) with a third reagent to form sample mixture 3.

[0099] In some embodiments, the continuous dilution mechanism or structure described herein includes: a first chamber configured for mixing a sample with a first reagent to form a sample mixture 1; a metering chamber connected to the first chamber and configured for metering a first portion of the sample mixture 1, wherein the metering chamber has a hydrophilic surface and is connected to a drive mechanism configured for driving the sample mixture 1 to contact the hydrophilic surface; and a second chamber connected to the metering chamber and configured for mixing the metered portion of the sample mixture 1 with a second reagent to form a sample mixture 2. In some embodiments, the drive mechanism is a pneumatic drive mechanism. In various embodiments, the continuous dilution mechanism or structure further includes a third chamber connected to the first chamber and configured for mixing a second portion of the sample mixture 1 with a third reagent to form a sample mixture 3.

[0100] In some embodiments, the third reagent is a dried or dehydrated reagent. In other embodiments, the third reagent is a liquid reagent. In some embodiments, the third reagent is a liquid reagent, and the second portion of sample mixture 1 is all the remaining volume of sample mixture 1 after its first portion has been removed.

[0101] In various embodiments, the fluid cartridge also includes a reagent storage unit.

[0102] In various embodiments, the reagent storage unit includes a storage chamber configured to contain a fluid reagent and a valve with a breakable seal. In some embodiments, the reagent storage unit further includes a vent with a breakable seal. In various embodiments, the reagent storage unit is connected to or also receives a pneumatically actuated mechanism to release the reagent contained therein. In various embodiments, the released reagent is used for continuous dilution. According to this disclosure, the reagent storage unit and / or a portion thereof includes a water vapor barrier material. In various embodiments, at least a portion of the storage chamber includes a water vapor barrier material. In various embodiments, the water vapor barrier material has a density of about 0.0001 g·mm / (m²). 2 ·day) to 0.01g·mm / (m 2 ·day), 0.01g·mm / (m 2 ·day) to 0.1g·mm / (m 2 ·day), 0.1g·mm / (m 2 ·day) to 1g·mm / (m 2 ·day) or 1g·mm / (m 2 ·day) to 5g·mm / (m 2 Water vapor permeability (day) at 23°C and 85% relative humidity.

[0103] In various embodiments, the fluid cartridge described herein includes at least two reagent storage units. In some embodiments, at least one reagent storage unit is used for a first reagent, and at least another reagent storage unit is used for a second reagent. In various embodiments, each of the at least two reagent storage units includes a storage chamber configured to contain a fluid reagent and a valve with a breakable connection. In various embodiments, when the fluid cartridge is received in a reader, the valves in the at least two reagent storage units open simultaneously.

[0104] In various embodiments, the fluid cartridge described herein also includes a chamber, a valve, a fluid passage, or a filter, or a combination thereof.

[0105] In various embodiments, the fluid cartridge described herein also includes regions for additional measurements (e.g., hemoglobin and hematocrit measurements). In various embodiments, the fluid cartridge described herein also includes an optical window for transmittance measurements. In some embodiments, transmittance measurements are used to analyze hemoglobin and / or hematocrit.

[0106] In various embodiments, the apparatus described herein also includes a reader configured to receive a fluid cartridge and perform cell measurements and / or analysis.

[0107] In various embodiments, the reader also includes a drive mechanism and / or a gate for shielding ambient light.

[0108] In various embodiments, the fluid cartridge received by the reader is positioned such that gravity pulls the fluid within the chamber away from the chamber vent and toward the bottom of the chamber. In various embodiments, the chamber is one or more of a first chamber, a second chamber, a third chamber, and a storage chamber. In some embodiments, the chamber is configured to store a fluid reagent. In this orientation, a gas gap is formed between the vent and the fluid reagent. Simultaneously, a channel accesses the chamber below the upper surface of the fluid reagent.

[0109] In various embodiments, the fluid cartridge received by the reader is positioned such that gravity pulls the fluid within the chamber away from the chamber vent and toward the bottom of the chamber. In various embodiments, the chamber is one or more of a first chamber, a second chamber, a third chamber, and a storage chamber. In some embodiments, the chamber is configured to form a diluted sample mixture. In this orientation, a gas gap is formed between the vent and the diluted sample mixture. Simultaneously, a channel enters the chamber below the upper surface of the diluted sample mixture. In some embodiments, the diluted sample mixture is transferred out of the chamber and enters a flow chamber via this channel to form a sample flow for cell counting analysis.

[0110] In various embodiments, this disclosure provides a serial dilution mechanism or structure. In various embodiments, the serial dilution mechanism or structure is part of a fluid cartridge for sample analysis (e.g., cell counting analysis). In various embodiments, this disclosure provides an apparatus including a fluid cartridge that incorporates a serial dilution mechanism or structure for sample analysis (e.g., cell counting analysis).

[0111] In various embodiments, the continuous dilution mechanism or structure described herein includes: a first chamber configured to mix a sample (e.g., a blood sample) with a first reagent to form a sample mixture 1; and a metering chamber connected to the first chamber and configured to meter a specified volume of the sample mixture 1. In various embodiments, the metering chamber has a hydrophilic surface and is connected to a drive mechanism configured to drive the sample mixture 1 to contact the hydrophilic surface. In some embodiments, the drive mechanism is a pneumatic drive mechanism. In various embodiments, the continuous dilution mechanism or structure further includes a second chamber connected to the metering chamber and configured to mix a metered portion of the sample mixture 1 with a second reagent to form a sample mixture 2. In various embodiments, the continuous dilution mechanism or structure further includes a third chamber connected to the first chamber and configured to mix a portion of the sample mixture 1 with a second reagent to form a sample mixture 3.

[0112] In some embodiments, the continuous dilution mechanism or structure further includes another metering chamber configured for metering a portion of sample mixture 1 used to form sample mixture 3. In some embodiments, the continuous dilution mechanism or structure is configured for mixing a metered volume of sample mixture 1 with a third reagent to form sample mixture 3. In other embodiments, the continuous dilution mechanism or structure is configured for mixing all remaining volumes of sample mixture 1 (after the first metered portion of sample mixture 1 has been removed) with a third reagent to form sample mixture 3.

[0113] In various embodiments, the fluid cartridge further includes a flow chamber connected to the first chamber and configured to form a sample flow of a sample mixture.

[0114] In various embodiments, this disclosure provides an apparatus including a fluid cartridge and a flow chamber, the fluid cartridge including a serial dilution mechanism or structure, and the flow chamber for cell counting analysis. In various embodiments, the serial dilution mechanism or structure includes: a first chamber configured for mixing a sample with a first reagent to form a sample mixture 1; a metering chamber connected to the first chamber and configured for metering a first portion of the sample mixture 1; and a second chamber connected to the metering chamber and configured for mixing the metered portion of the sample mixture 1 with a second reagent to form a sample mixture 2. In various embodiments, the flow chamber is connected to the first chamber and configured for forming a sample stream from any sample mixture and for measuring a signal from the sample stream to detect cells. In various embodiments, the metering chamber has a hydrophilic surface and is connected to a driving mechanism configured for driving the sample mixture 1 to contact the hydrophilic surface. In some embodiments, the driving mechanism is a pneumatic driving mechanism. In various embodiments, the serial dilution mechanism or structure further includes a third chamber connected to the first chamber and configured for mixing a second portion of the sample mixture 1 with a third reagent to form a sample mixture 3.

[0115] In various embodiments, the signal of the sample stream is measured to detect cells. In various embodiments, the flow chamber is configured to form two separate sample streams from sample mixture 2 and sample mixture 3, and the signals of the two sample streams are measured to detect cells. In some embodiments, the measurement sequence is: first sample mixture 2, then sample mixture 3. In some embodiments, the apparatus described herein is configured to introduce at least one gas gap between the sample mixtures being measured (e.g., sample mixture 2 and sample mixture 3).

[0116] In some embodiments, the third reagent is a drying or dehydrating reagent. In other embodiments, the third reagent is a liquid reagent. In some embodiments, the third reagent is a liquid reagent, and the second portion of sample mixture 1 is all the remaining volume of sample mixture 1 after its first portion has been removed.

[0117] In some embodiments, the flow chamber is a sheathless flow chamber, wherein the sample stream flows through the flow chamber without a sheath, and the sample stream has a diameter equal to or close to the diameter of the flow chamber. In some embodiments, the sheathless flow chamber has a width ranging from about 1 μm to 10 μm, 10 μm to 40 μm, 40 μm to 100 μm, or 100 μm to 200 μm, and a depth ranging from about 1 μm to 10 μm, 10 μm to 40 μm, 40 μm to 100 μm, or 100 μm to 200 μm. In some embodiments, the flow chamber is configured to use a sheath flow to narrow the diameter of the sample stream to a size smaller than the diameter of the flow chamber itself.

[0118] In various embodiments, the flow chamber includes an optically transparent window configured for measuring optical signals from the sample stream. Examples of optical signals include, but are not limited to, fluorescence, light scattering, light absorption, and light dissipation, and combinations thereof.

[0119] In various embodiments, the first and / or second reagents include a diluent, and the sample mixture 2 is measured to analyze red blood cells and / or platelets. In various embodiments, the third reagent includes a red blood cell lysis compound, and the sample mixture 3 is measured to analyze white blood cells or hemoglobin or both.

[0120] In various embodiments, the fluid cartridge also includes a reagent storage unit.

[0121] In various embodiments, the reagent storage unit includes a storage chamber configured to contain a fluid reagent and a valve with a breakable seal. In some embodiments, the reagent storage unit further includes a vent with a breakable seal. In various embodiments, the reagent storage unit is connected to or also receives a pneumatic actuation mechanism to release the reagent contained therein. In various embodiments, the released reagent is used for continuous dilution. According to this disclosure, the reagent storage unit and / or a portion thereof includes a water vapor barrier material. In various embodiments, at least a portion of the storage chamber includes a water vapor barrier material. In various embodiments, the water vapor barrier material has a density of about 0.0001 g·mm / (m²). 2 ·day) to 0.01g·mm / (m 2 ·day), 0.01g·mm / (m 2 ·day) to 0.1g·mm / (m 2 ·day), 0.1g·mm / (m 2 ·day) to 1g·mm / (m 2 ·day) or 1g·mm / (m 2 ·day) to 5g·mm / (m 2 Water vapor permeability (day) at 23°C and 85% relative humidity.

[0122] In various embodiments, the fluid cartridge described herein includes at least two reagent storage units. In some embodiments, at least one reagent storage unit is used for a first reagent, and at least another reagent storage unit is used for a second reagent. In various embodiments, each of the at least two reagent storage units includes a storage chamber configured to contain a fluid reagent and a valve with a breakable connection. In various embodiments, when the fluid cartridge is received into the reader, the valves in the at least two reagent storage units open simultaneously.

[0123] In various embodiments, the fluid cartridge described herein also includes a chamber, a valve, a fluid passage or a filter, or a combination thereof.

[0124] In various embodiments, the fluid cartridge described herein also includes regions for additional measurements (e.g., hemoglobin and hematocrit measurements). In various embodiments, the fluid cartridge described herein also includes an optical window for transmittance measurements. In some embodiments, transmittance measurements are used to analyze hemoglobin and / or hematocrit.

[0125] In various embodiments, the apparatus described herein also includes a reader configured to receive a fluid cartridge and perform cell measurements and / or analysis.

[0126] In various embodiments, the reader also includes a drive mechanism and / or a gate to block ambient light.

[0127] In various embodiments, the fluid cartridge received by the reader is positioned such that gravity pulls the fluid reagent within the chamber away from the chamber vent and toward the bottom of the chamber. In various embodiments, the chamber is one or more of a first chamber, a second chamber, a third chamber, and a storage chamber. In some embodiments, the chamber is configured to store the fluid reagent. In this orientation, a gas gap is formed between the vent and the fluid reagent. Simultaneously, a channel accesses the chamber below the upper surface of the fluid reagent.

[0128] In various embodiments, the fluid cartridge received by the reader is positioned such that gravity pulls the fluid reagent within the chamber away from the chamber vent and toward the bottom of the chamber. In various embodiments, the chamber is one or more of a first chamber, a second chamber, a third chamber, and a storage chamber. In some embodiments, the chamber is configured to form a diluted sample mixture. In this orientation, a gas gap is formed between the vent and the diluted sample mixture. Simultaneously, a channel enters the chamber below the upper surface of the diluted sample mixture. In some embodiments, the diluted sample mixture is transferred out of the chamber and enters a flow chamber via this channel to form a sample flow for cell counting analysis.

[0129] In various embodiments, this disclosure provides a method of using a reagent storage unit in a fluid cartridge, wherein the reagent storage unit includes a storage chamber configured to contain a fluid reagent and a valve with a breakable coupling. The method includes: storing the fluid reagent in the reagent storage unit; and applying pneumatic pressure to the reagent storage unit to transfer the fluid reagent out of the storage chamber. In various embodiments, the method described herein further includes applying an actuation mechanism to the fluid cartridge to open the breakable coupling of the valve. In various embodiments, the fluid reagent is transferred out of the storage chamber via the opened valve.

[0130] According to this disclosure, the reagent storage unit and / or a portion thereof includes a water vapor barrier material. In various embodiments, at least a portion of the storage chamber includes the water vapor barrier material. In various embodiments, the water vapor barrier material has a density of about 0.0001 g·mm / (m²). 2 ·day) to 0.01g·mm / (m 2 ·day), 0.01g·mm / (m 2 ·day) to 0.1g·mm / (m 2 ·day), 0.1g·mm / (m 2 ·day) to 1g·mm / (m 2 ·day) or 1g·mm / (m 2 ·day) to 5g·mm / (m 2 Water vapor permeability (day) at 23°C and 85% relative humidity.

[0131] In various embodiments, the reagent storage unit further includes a vent with a breakable seal. In various embodiments, pneumatic pressure is applied to the vent and opens the breakable seal to receive pneumatic pressure into the chamber.

[0132] In various embodiments, the method herein further includes receiving a sample (e.g., a blood sample) into a fluid cartridge to form a sample mixture with a fluid reagent.

[0133] In various embodiments, the methods described herein also include using a reader to receive a fluid cartridge therein for cell measurement and / or analysis.

[0134] In various embodiments, the received fluid cartridge is positioned such that gravity pulls the fluid reagent within the chamber away from the chamber vent and toward the bottom of the chamber. In various embodiments, the chamber is one or more of a first chamber, a second chamber, a third chamber, and a storage chamber. In some embodiments, the chamber is configured to form a diluted sample mixture. In this orientation, a gas gap is formed between the vent and the diluted sample mixture. Simultaneously, a channel enters the chamber below the upper surface of the diluted sample mixture. In some embodiments, the diluted sample mixture is transferred out of the chamber and enters a flow chamber via the channel to form a sample flow for cell counting analysis.

[0135] In various embodiments, the fluid cartridge described herein includes at least two reagent storage units. In some embodiments, at least one reagent storage unit is used for a first reagent, and at least another reagent storage unit is used for a second reagent. In various embodiments, each of the at least two reagent storage units includes a storage chamber configured to contain a fluid reagent and a valve with a breakable connection. In various embodiments, when the fluid cartridge is received in a reader, the valves in the at least two reagent storage units open simultaneously.

[0136] Figure 1A This document illustrates a non-limiting example of the dilution sequence in the sequential dilution method described herein. First, the sample is mixed with a first reagent (e.g., a liquid reagent or diluent) to form a single-diluted sample mixture 1. This sample can be a gaseous sample such as odor, a liquid sample such as bodily fluids (e.g., blood, lymph, sweat, tears, semen, saliva, and urine), and a solid sample such as nucleic acid extracts or tumor biopsies. A first portion of sample mixture 1 is metered and then mixed with a second reagent to form sample mixture 2. In some embodiments, the second reagent is a liquid reagent and sample mixture 2 is a secondary dilution from this sample. In other embodiments, the second reagent is a dried or dehydrated reagent. Additionally, a second portion of sample mixture 1 can be mixed with a third reagent to form sample mixture 3. In some embodiments, the third reagent is a liquid reagent. In other embodiments, the third reagent is a dried or dehydrated reagent. Sample mixture 2 and sample mixture 3 can be used for the same or different sample analyses. Various types of analytical techniques can be used to perform sample analysis, including but not limited to cell counting, spectroscopic methods (e.g., mass spectrometry, optical spectrometry, and ion mobility spectrometry), and chemiluminescence methods.

[0137] Figure 1B A non-limiting example of the dilution sequence in the sequential dilution method described herein is shown. First, a blood sample is mixed with a first reagent to form a sample mixture 1, which is a liquid diluent. Then, a first portion of sample mixture 1 is transferred to be mixed with a second reagent to form a sample mixture 2, which is also a liquid diluent. Simultaneously, a second portion of sample mixture 1 is transferred to be mixed with a third reagent to form sample mixture 3.

[0138] In all embodiments, the dilution rate of the sample mixture should be precisely controlled in the dilution sequence. This is particularly important for cell counting analysis that measures cell concentration.

[0139] In some embodiments, the dilution rate of sample mixture 1 is controlled by mixing a specified volume of a blood sample with a specified volume of a first reagent. In some embodiments, the dilution rate of sample mixture 2 is controlled by measuring a first portion of sample mixture 1 and mixing it with a specified volume of a second reagent. In some embodiments, the dilution rate of sample mixture 3 is controlled by measuring a second portion of sample mixture 1 and mixing it with a specified volume of a third reagent.

[0140] In some embodiments, the third reagent is a dried or dehydrated reagent, which is mixed with the second portion of sample mixture 1 to form sample mixture 3. Thus, the dilution rate of sample mixture 3 remains the same as or very close to the dilution rate of sample mixture 1, and the step of metering the second portion of sample mixture 1 is unnecessary.

[0141] In some embodiments, the total volume of sample mixture 1 is controlled by controlling a specified volume of the blood sample and a specified volume of the first reagent. A measured volume of sample mixture 1 is taken as a first portion to form sample mixture 2, and all remaining portions of sample mixture 1 are used as a second portion and mixed with a specified volume of a third reagent to form sample mixture 3. Thus, the dilution rate of sample mixture 3 can be precisely controlled without the need for a step of measuring the second portion of sample mixture 1.

[0142] This dilution order can be used for sample analysis (e.g., cell counting analysis, complete blood count test, and other measurements). As a non-limiting example, part or all of sample mixture 2 can be used for a first cell counting analysis (e.g., detection of red blood cells and platelets in a complete blood count test). As a non-limiting example, part or all of sample mixture 3 can be used for a second cell counting analysis (e.g., detection of white blood cells in a complete blood count test). As a non-limiting example, part or all of sample mixture 3 can be used for a third measurement (e.g., detection of hemoglobin concentration in a complete blood count test).

[0143] In complete blood count tests, high dilution rates (e.g., 500 to 2,000 times) are typically used for measuring red blood cells and platelets, while low or moderate dilution rates (e.g., 20 to 100 times) are used for measuring white blood cells and hemoglobin. Figure 1BIn this method, a blood sample is continuously diluted using a first liquid diluent followed by a second liquid diluent to achieve a high dilution rate in sample mixture 2 for the measurement of red blood cells and platelets; and when the third reagent is a dried or dehydrated reagent, sample mixture 3 is diluted once with liquid diluent to achieve a low or moderate dilution rate for the measurement of white blood cells and hemoglobin. As a non-limiting example, 10 μL of blood sample is diluted with 490 μL of the first reagent to form sample mixture 1 (total dilution rate 50-fold). 10 μL of sample mixture 1 is metered and transferred and diluted with 390 μL of the second reagent to form sample mixture 2 (total dilution rate 2,000-fold). Simultaneously, a portion of sample mixture 1 is transferred and mixed with the third reagent (e.g., a dried or dehydrated reagent) to form sample mixture 3 (total dilution rate 50-fold).

[0144] The total volume of liquid reagents used in this sequential dilution (e.g., 490 μL of the first reagent and 390 μL of the second reagent) is significantly less than the volume required for a single-step dilution (e.g., 19,990 μL of diluent for diluting a 10 μL blood sample 2,000-fold). This helps reduce the size of the fluidic cartridge device used to perform this dilution method. Furthermore, by sharing the dilution from the first step and using dried or dehydrated reagents to form the sample mixture, the complexity of the fluidic cartridge device for performing whole red blood cell count measurements is reduced.

[0145] Serial dilution sequences can also be used to measure parameters of sample mixtures for additional whole red blood cell counts. For example, the size of red blood cells or platelets can be measured in the first cell count analysis to determine hematocrit or plateletcrit. For instance, transmittance measurements can be performed on sample mixture 1, sample mixture 2, or both to determine hematocrit. In addition to parameters for whole red blood cell counts, serial dilution sequences can also be used for other cell count measurements, such as the ratio of helper / inducing T lymphocytes (CB4+) to suppressor / cytotoxic lymphocytes (CD8+).

[0146] Figure 2 A non-limiting example of a fluid diagram illustrating the sequential dilution method described herein is shown. A blood sample and a first reagent are introduced into chamber 201 to form sample mixture 1, and valves 211, 212, and 213 are closed to prevent sample mixture 1 from leaving the chamber. The chamber may have inlet ports for introducing the sample and the first reagent.

[0147] To collect a first portion of sample mixture 1, valve 211 is opened, and a portion of sample mixture 1 is transferred via fluid channel 221 into metering chamber 231, which collects a specified volume of sample mixture 1. The transfer of sample mixture 1 into metering chamber 231 is driven by an actuation mechanism. For example, fluid channel 221 and metering chamber 231 may have hydrophilic surfaces that introduce capillary forces to pull the sample mixture.

[0148] To form sample mixture 3, valve 212 is opened, and a portion of sample mixture 1 is transferred to chamber 203 via fluid channel 223. After the transfer is complete, valve 215 is closed to prevent the sample mixture from leaving the chamber. A third reagent is initially stored in chamber 203 and mixed with sample mixture 1 to form sample mixture 3. In some embodiments, the third reagent is a dried or dehydrated reagent. In some embodiments, the third reagent is a liquid reagent, and after the first portion is transferred to metering chamber 231, the entire remaining sample mixture 1 in chamber 201 is transferred as a second portion.

[0149] To form sample mixture 2, valve 214 is opened to mix the second reagent initially in chamber 202 with a first portion of sample mixture 1 collected in metering chamber 231 via fluid channel 222. As a non-limiting example, pneumatic pressure is applied to chamber 202 as an actuation mechanism, driving the second reagent to flow sample mixture 1 from metering chamber 231 into chamber 201, thereby forming sample mixture 2. In some embodiments, any residue of sample mixture 1 or sample mixture 3 remaining in chamber 201 is drained (e.g., into collection chamber 204) before sample mixture 2 is formed in chamber 201.

[0150] In various embodiments, a red blood cell lysis compound is introduced into chamber 201 before the first portion of sample mixture 1 and the second reagent are introduced into chamber 201 to form sample mixture 2. This step is important to minimize the impact of the residue of sample mixture 1 remaining in chamber 201 when analyzing sample mixture 2. For example, a single dilution of sample mixture 1 contains a much higher cell concentration (e.g., 400,000 red blood cells per microliter), while a further dilution of sample mixture 2 contains a much lower cell concentration (e.g., 10,000 red blood cells per microliter). Therefore, even a small volume (e.g., 1 microliter) of sample mixture 1 remaining in chamber 201 can have a significant impact on the analysis of sample mixture 2. Therefore, introducing a red blood cell lysis compound into the first chamber 201 to lyse the red blood cells in the residue of sample mixture 1 before forming sample mixture 2 in the first chamber 201 is beneficial for the analysis of sample mixture 2.

[0151] For the first cell count analysis, valve 213 is opened to allow at least a portion of sample mixture 2 to be transferred via fluid channel 224 into flow chamber 241, where sample mixture 2 forms a sample flow for measurement (e.g., detection of red blood cells and platelets in a complete blood count test). Any sample mixture leaving flow chamber 241 also flows into collection chamber 204 via fluid channel 225. In some embodiments, after the first cell count analysis, any residue of sample mixture 2 in chamber 201 is removed (e.g., enters collection chamber 204). For the second cell count analysis, sample mixture 3 in chamber 203 is first transferred to chamber 201, and then at least a portion of sample mixture 3 is transferred to flow chamber 241 for measurement (e.g., detection of white blood cells in a complete blood count test). Simultaneously, other measurements can be performed on the sample mixture. As a non-limiting example, a third measurement (e.g., detection of hemoglobin in a complete blood count test) is performed on sample mixture 3 in chamber 201 (e.g., using a transmittance detection method). As a non-limiting example, another measurement (e.g., detecting hematocrit in a complete blood count test) is performed on the sample mixture 1 in chamber 201 (e.g., using a method for detecting transmittance).

[0152] Various designs of the flow chamber 241 can be used for cell counting analysis. In flow cytometry, flow chamber designs typically utilize sheath flow to narrow the diameter of the sample stream to a size smaller than the diameter of the flow chamber itself. In some embodiments of this disclosure, such as Figure 3 As shown, a sheathless flow chamber design is used. In this design, the sample stream flows through the flow chamber without a sheath and has a diameter equal to or close to the diameter of the flow chamber. This sheathless design reduces the complexity of the fluid map for performing cell counting analysis in a sequential dilution sequence. Various types of signals used for cell counting analysis can be measured in the flow chamber. As a non-limiting example, incident light illuminates the sample stream in the flow chamber, and optical signals from the sample stream (including, but not limited to, fluorescence, light scattering, light absorption, and light dissipation, and combinations thereof) are measured simultaneously. Various flow chamber designs and types of signals detected are described in U.S. Patent No. 15 / 803,133 and International Application No. PCT / US17 / 59965, the entire contents of which are incorporated herein by reference as fully set forth herein.

[0153] exist Figure 2 In some embodiments of the fluid diagram, a third measurement (e.g., detecting hemoglobin in a complete blood count test) is performed on the sample mixture 3 in chamber 201. Various measurement signals and device designs can be used for this measurement. Figure 4The illustrated non-limiting example shows an optical path formed between two transparent surfaces and the transmittance along that path is measured. In this example, the two transparent surfaces (surface 1 and surface 2) are located on chamber 201. Incident light enters chamber 201 through surface 1, passes through the sample mixture within chamber 201, and exits chamber 201 through surface 2. The transmitted light behind surface 2 is measured. The transmittance, which is the ratio of the intensity of the incident light to the intensity of the transmitted light, is analyzed to determine the hemoglobin concentration. Various apparatus designs and methods for transmitting light are described in International Application No. PCT / US17 / 62765 and U.S. Patent No. 15 / 819,416, the entire contents of which are incorporated herein by reference as fully set forth herein.

[0154] Various reagents, or combinations thereof, can be used for serial dilutions to obtain measurements of complete blood cell counts. As a non-limiting example, the use of... Figure 2 The first reagent in the fluid diagram includes a dilution buffer, which dilutes the blood sample to minimize or avoid hemolysis of red blood cells. Examples of the first reagent include, but are not limited to, aqueous solutions of sodium chloride or potassium chloride, or phosphate-buffered saline, or equivalents thereof. The molar osmotic concentration of the first reagent is adjusted to minimize undesirable hemolysis, for example, at approximately 140 mOsm / L to 160 mOsm / L, 160 mOsm / L to 180 mOsm / L, 180 mOsm / L to 200 mOsm / L, 200 mOsm / L to 220 mOsm / L, 220 mOsm / L to 240 mOsm / L, and 240 mOsm / L to 260 mOsm / L. / L, within the range of 260mOsm / L to 280mOsm / L, 280mOsm / L to 300mOsm / L, 300mOsm / L to 320mOsm / L, 320mOsm / L to 340mOsm / L, 340mOsm / L to 360mOsm / L, 360mOsm / L to 380mOsm / L, or 380mOsm / L to 400mOsm / L. When mixed with blood samples, it forms molar osmotic concentrations of approximately 140 mOsm / L to 160 mOsm / L, 160 mOsm / L to 180 mOsm / L, 180 mOsm / L to 200 mOsm / L, 200 mOsm / L to 220 mOsm / L, 220 mOsm / L to 240 mOsm / L, 240 mOsm / L to 260 mOsm / L, and 26... 1. Sample mixtures with concentrations of 0 mOsm / L to 280 mOsm / L, 280 mOsm / L to 300 mOsm / L, 300 mOsm / L to 320 mOsm / L, 320 mOsm / L to 340 mOsm / L, 340 mOsm / L to 360 mOsm / L, 360 mOsm / L to 380 mOsm / L, or 380 mOsm / L to 400 mOsm / L.

[0155] In some embodiments, the first reagent further includes a fluorescent dye, such as a nucleic acid dye having a high affinity for binding to DNA, or RNA, or both DNA and RNA. The fluorescent dye labels leukocytes and platelets, distinguishing them from other particles of similar size in the sample mixture. The fluorescent signal can also be used to identify different leukocyte subtypes, such as lymphocytes, monocytes, neutrophils, eosinophils, or basophils. Examples of fluorescent dyes include, but are not limited to, propidium iodide, ethidium bromide, DAPI, Hoechst dye, acridine orange, thiazole orange, 7-AAD, LDS751, Basic Orange 21, Capri blue, Nile blue, Brilliant cresol blue, etc.

[0156] As a non-restrictive example, Figure 2 The second reagent in the fluid diagram includes a dilution buffer, which further dilutes the sample mixture 1 to minimize or avoid hemolysis of red blood cells. Examples of the second reagent include, but are not limited to, aqueous solutions of sodium chloride or potassium chloride, or phosphate-buffered saline, or equivalents thereof. Adjust the molar osmotic concentration of the second reagent to minimize undesirable hemolysis, for example, in the range of approximately 140-160 mOsm / L, 160-180 mOsm / L, 180-200 mOsm / L, 200-220 mOsm / L, 220-240 mOsm / L, 240-260 mOsm / L, 260-280 mOsm / L, 280-300 mOsm / L, 300-320 mOsm / L, 320-340 mOsm / L, 340-360 mOsm / L, 360-380 mOsm / L, or 380-400 mOsm / L. When mixed with sample mixture 1, it forms sample mixture 2 with a molar osmotic pressure concentration of approximately 140-160 mOsm / L, 160-180 mOsm / L, 180-200 mOsm / L, 200-220 mOsm / L, 220-240 mOsm / L, 240-260 mOsm / L, 260-280 mOsm / L, 280-300 mOsm / L, 300-320 mOsm / L, 320-340 mOsm / L, 340-360 mOsm / L, 360-380 mOsm / L, or 380-400 mOsm / L.

[0157] In some embodiments, the second reagent has a molar osmotic concentration of approximately 140-160 mOsm / L, 160-180 mOsm / L, 180-200 mOsm / L, 200-220 mOsm / L, 220-240 mOsm / L, 240-260 mOsm / L, 260-280 mOsm / L, or 280-300 mOsm / L. The resulting sample mixtures exhibited final molar osmotic concentrations of 140-160 mOsm / L, 160-180 mOsm / L, 180-200 mOsm / L, 200-220 mOsm / L, 220-240 mOsm / L, 240-260 mOsm / L, 260-280 mOsm / L, and 280-300 mOsm / L. These molar osmotic concentration levels introduce hypotonicity into the erythrocytes and contribute to increasing the transformation of erythrocytes from biconcave to spherical shapes. Consequently, it is possible to measure erythrocyte size with minimal variation in different cellular orientations.

[0158] In some embodiments, the second agent further includes a surfactant or salt that helps to significantly transform red blood cells from a biconcave shape to a spherical shape. Examples of surfactants or salts include, but are not limited to, sodium dodecyl sulfate, octyltrimethylammonium chloride, decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, trimethyltetradecylammonium chloride, hexadecyltrimethylammonium chloride, etc.

[0159] In some embodiments, the second reagent further includes a fluorescent dye, such as a nucleic acid dye having a high affinity for binding to DNA, or RNA, or both DNA and RNA. The fluorescent dye labels platelets and distinguishes them from other particles of similar size (e.g., lipid particles) in the sample mixture 2, thus improving the accuracy of platelet detection. Examples of fluorescent dyes include, but are not limited to, thiazole orange, capreon blue, Nile blue, Brilliant cresol blue, acridine orange, Basic Orange 21, etc.

[0160] In various embodiments, the fluorescent dye in the first reagent is introduced into both sample mixture 2 and sample mixture 3. In various embodiments, the fluorescent dye in the second reagent is introduced only into sample mixture 2. In some embodiments, the fluorescent dye in the first reagent and the fluorescent dye in the second reagent may be the same dye. In other embodiments, the fluorescent dye in the first reagent and the fluorescent dye in the second reagent may be different dyes, thereby optimizing the fluorescent labeling in sample mixture 2 and sample mixture 3, respectively.

[0161] As a non-restrictive example, Figure 2The third reagent in the fluid diagram includes a lysis compound that lyses red blood cells in sample mixture 3 to release hemoglobin. Examples of lysis compounds include, but are not limited to, ammonium salts, quaternary ammonium salts, pyridinium salts, hydroxylamine salts, nonionic surfactants, ionic surfactants, sodium dodecyl sulfate, sodium lauryl sulfate, etc.

[0162] In some embodiments, the third agent further includes a compound that helps dissolve the membrane of red blood cells or fragments of lysed red blood cells. Examples of compounds include, but are not limited to, surfactants such as BC30TX, polyoxyethylene hexadecyl ether, saponins, Tween 20, Triton X-100, etc. In some embodiments, the compound is a nonionic surfactant, such as BC30TX and saponins.

[0163] In some embodiments, the third reagent further includes a fluorescent dye, such as a nucleic acid dye having a high affinity for binding to DNA, or RNA, or both DNA and RNA. The fluorescent dye labels leukocytes to distinguish them from other particles of similar size in the sample mixture and introduces a fluorescent signal to identify different leukocyte subtypes, such as lymphocytes, monocytes, neutrophils, eosinophils, or basophils. Examples of fluorescent dyes include, but are not limited to, propidium iodide, ethidium bromide, DAPI, Hurst dye, acridine orange, thiazole orange, 7-AAD, LDS751, Basic Orange 21, etc.

[0164] In some embodiments, the third reagent is stored in a dried or dehydrated form before being mixed with sample mixture 1 to form sample mixture 3. Thus, the dilution rate of sample mixture 3 remains the same or very close to that of sample mixture 1. Therefore, no additional metrological steps are required to precisely control the dilution rate of sample mixture 3. This helps to simplify fluid mapping and device design (e.g., in a disposable fluid cartridge) for implementing whole blood cell count measurements.

[0165] In some embodiments, the third reagent is stored in the form of a thin layer comprising a dried coating of the reagent compound, which dissolves rapidly upon contact with sample mixture 1. The thickness of the dried coating is about 1,000 μm to 100 μm, 100 μm to 10 μm, 10 μm to 1 μm, 1 μm to 0.1 μm, 0.1 μm to 0.01 μm, or 0.01 μm to 0.001 μm. Various methods can be used to form the dried coating. As a non-limiting example, firstly, the reagent compound is dissolved in a solvent to form a liquid solution; then, the liquid solution is applied to a solid substrate (e.g., a plastic surface) on a chamber; finally, the solvent in the liquid solution is rapidly dried to coat the reagent compound onto the substrate.

[0166] Further examples of reagent compounds or combinations thereof are disclosed in International Application No. PCT / US17 / 62765 and U.S. Patent Application No. 15 / 819,416, the entire contents of which are incorporated herein by reference as fully set forth.

[0167] Figure 5A A non-limiting example of a fluid cartridge for performing serial dilutions in a complete blood count test is shown. In this fluid cartridge 500, a first reagent 581 is initially received in chamber 501, and a closed valve 521 prevents the reagent from leaving the chamber. An inlet orifice 517 receives a blood sample into the cartridge, and a fluid channel 552 further guides the blood sample into a fluid channel 553 and a metering chamber 533. In some embodiments, the surfaces of fluid channels 552 and 553 and the surface of metering chamber 533 are hydrophilic, providing capillary forces to draw the blood sample into the flow. When the blood sample reaches capillary valve 529, it stops flowing, and a designated volume of the sample is collected in metering chamber 533.

[0168] To mix the first reagent with the blood sample, valve 521 is opened, and an actuation mechanism is applied to reagent 581 in chamber 501. This actuation mechanism drives the first reagent into fluid channel 551, which connects to fluid channel 553 and metering chamber 533. The first reagent 581 causes the blood sample in fluid channel 553 and metering chamber 533 to flow through capillary valve 529 into fluid channel 554, and then into chamber 504 to form sample mixture 1. Valves 524, 525, 526, and 527 are then closed to prevent sample mixture 1 from leaving chamber 504. Various actuation mechanisms can be used to drive reagent 581. As a non-limiting example, when ambient atmospheric pressure is connected to vent 514 of chamber 504, a pneumatic pressure higher than ambient atmospheric pressure is applied to vent 511 of chamber 501. This pneumatic configuration uses pneumatic force to drive reagent 581 from chamber 501 to chamber 504.

[0169] To collect a portion of sample mixture 1 as a first part for mixing with a second reagent, valve 525 is opened and an actuation mechanism is applied to sample mixture 1 to drive it into fluid channel 558 and subsequently into metering chamber 532. Capillary valve 528 stops the flow of sample mixture and collects a specified volume of the sample mixture in metering chamber 532. Various actuation mechanisms can be used for the transfer of the sample mixture. As a non-limiting example, vent 516 is configured to connect to metering chamber 532 via fluid channels 556 and 557. When ambient atmospheric pressure is connected to vent 514 of chamber 504, a pneumatic pressure below ambient atmospheric pressure is applied to vent 516. This configuration applies pneumatic force to pull sample mixture 1 from chamber 504 into fluid channel 558 and subsequently into metering chamber 532. In some embodiments, at least a portion of the surface of metering chamber 532 (e.g., surface area 534, colored gray in the figure) is hydrophilic. When sample mixture 1 flows to contact surface region 534, the initial driving mechanism is removed. The hydrophilic surface of region 534 introduces a capillary force that continues to pull sample mixture 1 into the metering chamber. This flow stops when it reaches capillary valve 528.

[0170] To form sample mixture 3, valve 524 is opened and an actuation mechanism is applied to the sample mixture 1 remaining in chamber 504. This actuation mechanism drives a second portion of sample mixture 1 into fluid channel 559 and subsequently into chamber 503. Various actuation mechanisms can be used for the transfer of this sample mixture. As a non-limiting example, when ambient atmospheric pressure is connected to vent 514 of chamber 504, a pneumatic pressure below ambient atmospheric pressure is applied to vent 513 of chamber 503. Once the second portion of sample mixture 1 has been driven into chamber 503, valve 523 can be closed. A third reagent is initially received in chamber 503. In some embodiments, the third reagent 583 is stored in chamber 503 in the form of a dried or dehydrated reagent or a dehydrated coating. The portion of sample mixture 1 transferred to chamber 503 dissolves at least a portion of the dried or dehydrated reagent or the dehydrated coating, forming sample mixture 3. In some embodiments, the third reagent 583 is stored in chamber 503 in the form of a liquid reagent, and after the first portion is transferred to metering chamber 532, the entire remaining sample mixture 1 in chamber 504 is transferred as the second portion.

[0171] In some embodiments, any remaining sample mixture in chamber 504 is driven out of the chamber (e.g., into collection chamber 505) before the formation of sample mixture 2 continues. In some embodiments, a second reagent 582 is initially received in chamber 502 and stored in liquid form. To form sample mixture 2, valve 522 is opened and vent 516 is closed (e.g., via an external seal). A driving mechanism is applied to the second reagent 582, which flows into fluid channel 555, and consequently causes sample mixture 1 in metering chamber 532 to flow into chamber 504 and form sample mixture 2. As a non-limiting example of the driving mechanism, when vent 514 is connected to ambient atmospheric pressure, a pneumatic pressure below ambient atmospheric pressure is applied to vent 512.

[0172] For the first cell count analysis, valve 527 is opened and a driving mechanism is applied to the sample mixture 2 in chamber 504. At least a portion of the sample mixture 2 is driven into fluid channel 560 and then into flow chamber 531, forming a sample stream for the first cell count analysis. In some embodiments, the sample stream exiting flow chamber 531 flows through fluid channel 561 and is received into collection chamber 505. As a non-limiting example of the driving mechanism, when vent 514 is connected to ambient atmospheric pressure, a pneumatic pressure lower than ambient atmospheric pressure is applied to vent 515 of chamber 505.

[0173] In a complete blood count test, a first cell count analysis detects red blood cells, platelets, or both in the sample mixture 2. Different types of signals (e.g., optical signals, electrical signals, or combinations of both) can be measured in the flow cell 531 for detection. In some embodiments, the detection of red blood cells and platelets uses at least an optical signal, and the flow cell 531 includes a transparent window for optical measurement. Examples of optical signals include, but are not limited to, fluorescence, light scattering, light absorption, and light dissipation, and combinations thereof. In some embodiments, the first cell count analysis determines the number of red blood cells, platelets, or both in the sample. In some embodiments, the measurement also determines other characteristics of the red blood cells or platelets, including, but not limited to, the size of the red blood cells or platelets.

[0174] For the second cell count analysis, sample mixture 3 is first transferred to chamber 504 and then driven to flow chamber 531 to form a sample stream (e.g., by the same or similar driving mechanism used in the first cell count analysis). In some embodiments, any remaining sample mixture in chamber 504 is transferred out (e.g., into collection chamber 505) before sample mixture 3 is transferred to chamber 504. The second cell count analysis of the sample stream detects white blood cells in sample mixture 3. Different types of signals (e.g., optical signals, electrical signals, or combinations of both) can be measured in flow chamber 531. In some embodiments, the detection of white blood cells uses at least an optical signal. Examples of optical signals include, but are not limited to, fluorescence, light scattering, light absorption, and light extinction, and combinations thereof. In a complete blood count test, the second cell count analysis determines the number of white blood cells in the sample. In some embodiments, the analysis also determines the number of white blood cell subtypes, such as lymphocytes, monocytes, neutrophils, eosinophils, or basophils.

[0175] In addition to the first and second cell count analyses, other measurements can be performed on the sample mixture. In some embodiments, a third measurement is performed on the sample mixture 3 in chamber 504 to detect hemoglobin in the sample. Various types of signal and device designs can be used for this measurement, and non-limiting examples using light transmittance measurements have been provided. Figure 4 As shown in the diagram. In some embodiments, a third measurement of hemoglobin is performed on the sample mixture 3 prior to the second cell count analysis. In other embodiments, a third measurement is performed on a portion of the sample mixture 3 remaining in chamber 504 after the second cell count analysis.

[0176] In some embodiments, additional measurements are also performed on one or more of the sample mixtures used for complete blood cell counting. In a non-limiting example, measurements are performed on sample mixture 1, or sample mixture 2, or both. Figure 4 The light transmittance measurement shown is used to determine hematocrit in a sample. Various methods using light transmittance measurement, as described in U.S. Patent No. 6,064,474, the entire contents of which are incorporated herein by reference as if set forth herein in their entirety.

[0177] exist Figure 5AIn a non-limiting example, both cell counting analysis of sample mixture 2 and cell counting analysis of sample mixture 3 are performed in the same flow chamber 531. The order in which the two sample mixtures are measured is interchangeable. In some embodiments, sample mixture 2 is measured in the flow chamber before sample mixture 3, and this order can reduce the risk of unwanted bubble formation in the flow chamber. This is because sample mixture 2 has a higher dilution rate than sample mixture 3, thus reducing the protein concentration from the blood sample. Proteins adhering to the flow chamber can alter the surface properties of the flow chamber and make it easier to form unwanted bubbles. In other embodiments, sample mixture 3 is measured in the flow chamber before sample mixture 2. In some embodiments, at least a section of gas is pumped into the flow chamber between the measurement of sample mixture 2 and the measurement of sample mixture 3. The gas gap helps to provide separation between the two sample streams.

[0178] Various types of valves (e.g., passive or active valves) can be used as valves 521, 522, 523, 524, 525, 526, and 527. In some embodiments, passive valves are used. Figures 6A-6F A non-limiting example of a passive valve is shown. Figure 6A The valve in the design comprises a channel with a hydrophobic surface and a junction where the channel diameter rapidly narrows. When fluid flows to the junction, the sudden increase in capillary force at the junction stops the flow and acts as a closed valve. To open the valve, an actuating mechanism (e.g., such as...) is activated. Figure 6B The pneumatic pressure shown is applied to the fluid and pushes it through the joint and into the downstream channel. Figure 6C The valve in the design includes a channel with a hydrophilic surface and a junction where the channel diameter rapidly increases. When fluid flows to this junction, the sudden decrease in capillary force at the junction acts as a closed valve, stopping the flow. To open the valve, an actuating mechanism (e.g., such as...) is activated. Figure 6D The pneumatic pressure shown is applied to the fluid and pushes it through the joint and into the downstream channel. Figure 6C The valve operates as a check valve, which stops the flow of fluid from the narrower side of the joint to the wider side. Figure 6E and Figure 6F A non-limiting example of a two-way valve is shown, which includes a hydrophobic patch in the channel. When fluid flows to the hydrophobic patch, the capillary force of the hydrophobic surface acts as a closed valve, stopping the fluid flow. Figure 6E and 6F As shown, this valve design stops the flow in both directions of the channel.

[0179] Various types of valves can be used as capillary valves 528 and 529. In some embodiments, passive valves are used as capillary valves. Figures 6A-6FA non-limiting example of a passive valve as described herein is shown.

[0180] Figure 5B Another non-limiting example of a fluid cartridge implementing a continuous dilution method is shown. When active valves are used as valves 521 and 522, this design is similar to... Figure 5A The example is similar. The design also includes two actuation structures, actuation structure 591 and actuation structure 592, which are used to open valves 521 and 522, respectively. The operation of this design is also similar to... Figure 5A Similar examples exist. More options are introduced for the function of the fluid cartridge by using active valves. In some embodiments, a first reagent 581 and a second reagent 582 are initially received in chambers 501 and 502, and valves 521 and 522 are initially closed to store the reagents in those chambers, respectively.

[0181] Various timings for opening valves 521 and 522 can be used for serial dilutions to perform sample analysis (e.g., cell counting analysis and complete blood count tests). In some embodiments, valves 521 and 522 are opened independently. In other embodiments, valves 521 and 522 are opened simultaneously. As a non-limiting example, valves 521 and 522 are opened simultaneously, for example, by an actuation module in the reader, when the fluid cartridge is received in the reader for a complete blood count test. By opening both valves in the same step, the actuation mechanism for opening the valves is simplified. When both valves are opened simultaneously, different strategies for fluid transfer can be used. As a non-limiting example, vent 514 is constantly connected to ambient atmospheric pressure when pneumatic pressure is applied independently to orifices 511 and 512. By applying pneumatic pressure to orifice 511, reagents or sample mixtures are driven to transfer between chambers 501 and 504. By applying pneumatic pressure to orifice 512, a reagent or sample mixture is driven to transfer between chambers 502 and 504. These two transfer steps are independently controlled by pneumatic pressure at orifices 511 and 512. Various strategies can be used for fluid transfer, and non-limiting examples of using pneumatic pressure to control fluid transfer are described in patent applications PCT / US17 / 59965, 15 / 803,133, PCT / US17 / 62765, 15 / 819,416, 62 / 575,918, and PCT / US18 / 56725, the entire contents of which are incorporated herein by reference as fully set forth.

[0182] Various types of active valves can be used as valves 521 and 522. In some embodiments, valves with a breakable connection are used. Figure 7A (A cross-sectional view of the fluid cell 500 along line AA′) is shown in Figure 5BA non-limiting example of an active valve with a breakable bond is implemented in a fluid cartridge 500. In this example, valve 521 includes a breakable bond 703 that initially bonds a flexible membrane 701 to a solid wall 704 surrounding chamber 501. When this bond is intact, valve 521 prevents a first reagent 581 in chamber 501 from leaving and entering fluid passage 551. Figure 7B As shown, by applying an actuation mechanism (e.g., deformation of the flexible membrane 702 supporting the actuation structure 591), the actuation structure 591 pushes the membrane 701 to break the bond 703 and form a fluid channel 707. A driving mechanism applied to the first reagent 581 (e.g., pneumatic pressure applied to the vent 511) can transfer the reagent from the chamber 501 to the fluid channel 551 via the fluid channel 707. Various methods can be used to form a breakable bond. One non-limiting example is a bond formed by a layer of adhesive material. The adhesive layer bonds the membrane 701 to the solid wall 704. When the membrane 701 has sufficiently large deformation, the adhesive layer separates from the membrane 701 or the solid wall 704, thereby breaking the bond. Various methods can be used to provide the actuation mechanism. One non-limiting example is applying a mechanical thrust to the membrane 702 to introduce deformation.

[0183] In some embodiments, a reagent storage unit is constructed within a fluid cartridge for storing reagents within the cartridge. Figure 7C A non-limiting example is shown, comprising a chamber 501, a solid wall 704 forming the chamber, membranes 701 and 702, a valve 521 with a breakable seal 703, a vent 511, and a breakable seal 705 closing the vent 511. After reagent 581 is received into chamber 501, seal 705 is applied to close vent 511. Seal 705, valve 521, membranes 701 and 702, and solid wall 704 make chamber 501 a completely sealed container for storing reagent 581. By using materials with high water vapor barrier properties for the seals, valves, membranes, and solid walls, the sealed container minimizes water vaporization, thereby minimizing volume loss of the stored reagent. Examples of such materials include, but are not limited to, aluminum foil and plastic films with low water permeability (e.g., cyclic olefin polymers, cyclic olefin copolymers, polyvinyl chloride, and high-density polyethylene). Thus, a specified volume of reagent received into the chamber can be stored for an extended period without significant volume loss.

[0184] The seal 705 can be opened at any time before or after the cartridge is received by the reader for measurement. In a non-limiting example, the seal 705 is opened by mechanical puncture after the fluid cartridge is received by the reader. To transfer reagent 581 out of chamber 501, valve 521 is opened by breaking the coupling 703, and an actuation mechanism is applied to the reagent to drive it into fluid channel 551 for continuous dilution. In some embodiments, pneumatic pressure is applied to vent 511 as the reagent actuation mechanism. Figure 8C As shown, by positioning the chamber in a vertical or slightly inclined position, gravity constantly pulls the reagent toward the bottom of chamber 501. Furthermore, by positioning the fluid channel 551 near the bottom of the chamber, a driving mechanism (e.g., pneumatic pressure) can push reagent 581 out of the chamber and into the fluid channel 551. Thus, a specified volume of reagent 581 can be transferred for continuous dilution without significant dead volume.

[0185] Therefore, in this storage unit, a specified volume of reagent can be stored long-term without significant volume loss and can also be transferred for serial dilution without significant dead volume. Using this design, there is no need for additional measurement of reagent volume to ensure the target dilution rate. Thus, the storage unit simplifies the cartridge design for implementing serial dilutions. In various embodiments, the storage unit is used to store reagents in volumes ranging from about 10 μL to 50 μL, 50 μL to 100 μL, 100 μL to 200 μL, 200 μL to 500 μL, 500 μL to 1,000 μL, or 1,000 μL to 5,000 μL.

[0186] This reagent storage unit can be used in fluid cartridges for implementing the serial dilution method as described herein, as well as in fluid cartridges for implementing any other fluid function. Various other types of reagent storage units can also be used for fluid cartridges implementing the serial dilution method as described herein. Non-limiting examples of other types of reagent storage units are described in U.S. Patent Application Nos. 62 / 504,866 and PCT / US18 / 31893, the entire contents of which are incorporated herein by reference as if fully set forth.

[0187] In various embodiments, the blood sample received in the fluid cartridge device for measurement has a volume ranging from about 0.01 μL to 0.1 μL, 0.1 μL to 1 μL, 1 μL to 10 μL, or 10 μL to 100 μL. In various embodiments, a specified volume of the received blood sample is mixed with a first reagent to form sample mixture 1, and the specified volume is about 10% to 40%, 40% to 80%, 80% to 90%, 90% to 99%, or 99% to 100% of the volume of the received blood sample. In various embodiments, the first reagent has a volume ranging from about 10 μL to 50 μL, 50 μL to 100 μL, 100 μL to 200 μL, 200 μL to 500 μL, or 500 μL to 1,000 μL. In various embodiments, sample mixture 1 has a dilution ratio ranging from about 1:5 to 1:10, 1:10 to 1:20, 1:20 to 1:50, or 1:50 to 1:100 (i.e., the volume of the blood sample being mixed versus the volume of the first reagent being mixed). In various embodiments, a specified volume of sample mixture 1 is metered to mix with a second reagent to form sample mixture 2. In various embodiments, the metered volume of sample mixture 1 ranges from about 0.1 μL to 1 μL, 1 μL to 10 μL, or 10 μL to 100 μL. In various embodiments, the second reagent has a volume ranging from about 10 μL to 50 μL, 50 μL to 100 μL, 100 μL to 200 μL, 200 μL to 500 μL, or 500 μL to 1,000 μL. In various embodiments, sample mixture 2 has a dilution ratio ranging from about 1:5 to 1:10, 1:10 to 1:20, 1:20 to 1:50, or 1:50 to 1:100 (i.e., the volume of sample mixture 1 being measured: the volume of the second reagent being mixed). In various embodiments, when a fluid reagent is used as a third reagent, it has a volume ranging from about 0.1 μL to 1 μL, 1 μL to 10 μL, 10 μL to 100 μL, 100 μL to 200 μL, 200 μL to 500 μL, or 500 μL to 1,000 μL.

[0188] In some embodiments, a filter structure is added to the fluid cartridge 500 to prevent aerosols or liquids from leaving the cartridge. Figure 7D In a non-limiting example, a filter 706 is added between chamber 501 and vent 511. The filter 706 comprises a porous material that allows air to pass through but prevents aerosols or liquids from entering. Examples of porous materials include, but are not limited to, Porex, porous polyethylene, porous polytetrafluoroethylene, Pall's Versapor R membrane, and Sterlitech's Aspire microfiltration membrane.

[0189] In some embodiments, such as Figure 5CIn the non-limiting example shown, multiple valves can be used in combination as valve components in the fluid cartridge 500. This design is consistent with... Figure 5A and Figure 5B The design is similar, but with the following differences: it uses an active valve 521 plus a passive valve 593 for the valve assembly of chamber 501, and an active valve 522 plus a passive valve 594 for the valve assembly of chamber 502. The additional passive valves 593 and 594 help simplify fluid transfer within the cartridge. For example, when the fluid cartridge is received into the reader, the active valve 521 is opened, and the passive valve 593 prevents the first reagent 581 from entering the fluid channel 551 before the actuation mechanism is applied.

[0190] Figure 5D A non-limiting example of a fluid structure for metering a first portion of sample mixture 1 is shown, which can be used for serial dilution and sample analysis. The fluid structure includes: a first chamber 504 configured to receive a sample and a first reagent to form sample mixture 1; and a metering chamber 532. Metering chamber 532 is connected to the first chamber 504 via a fluid channel 558 and configured to meter a specified volume of sample mixture 1. The fluid structure also includes a vent 514 connected to the first chamber 504 and a vent 516 connected to the metering chamber 532. In various embodiments, the surfaces of the fluid channel 558 and the metering chamber 532 may be hydrophilic to provide capillary forces to drive sample mixture 1 into the metering chamber 532. In some embodiments, the surface of metering chamber 532 may be hydrophilic. In some embodiments, a portion of the surface of metering chamber 532 may be hydrophilic (e.g., surface region 534, colored gray in the figures). In some embodiments, the hydrophilic surface described herein may be made using an inherently hydrophilic material. In other embodiments, the hydrophilic surface described herein can be fabricated by applying a hydrophilic coating to a non-hydrophilic surface. In various embodiments, the metering chamber 532 also includes a capillary valve 528 to stop the flow of the sample mixture 1 driven by capillary forces.

[0191] To collect a first portion of sample mixture 1 from the first chamber 504, a driving mechanism is applied to the fluid structure to drive sample mixture 1 out of the first chamber 504 and into the metering chamber 532. In various embodiments, the driving mechanism is a pneumatic force. As a non-limiting example of applying a pneumatic force, vent 516 is connected to a pressure below ambient atmospheric pressure, and vent 514 is connected to ambient atmospheric pressure. As another non-limiting example of applying a pneumatic force, vent 516 is connected to ambient atmospheric pressure, and vent 514 is connected to a pressure above ambient atmospheric pressure. In various embodiments, the driving mechanism can be other types of force. Non-limiting examples of other types of force include, but are not limited to, gravity, capillary force, electrophoresis, magnetic force, acoustic pressure, and centrifugal force, and combinations thereof.

[0192] Driven by the application-driven mechanism, sample mixture 1 leaves the first chamber 504 and enters the metering chamber 532. For example... Figure 5E As shown, the driving mechanism is removed after the sample mixture 1 contacts the hydrophilic surface region 534 in the metering chamber 532. After the driving mechanism is removed, the sample mixture 1, driven by capillary forces, continues to flow and fill the metering chamber 532. Figure 5F As shown, the flow of sample mixture 1 is stopped at capillary valve 528, so a first portion of sample mixture 1 with a specified volume is collected.

[0193] Figure 5G A non-limiting example of a fluid structure for implementing serial dilution is shown, which is used in various sample analysis methods. The fluid structure includes a first chamber 504 configured to receive a sample and a first reagent to form a sample mixture 1. The fluid structure also includes a metering chamber 532 connected to the first chamber 504 via a fluid channel 558 and configured to meter a specified volume of the sample mixture 1. The fluid structure also includes a second chamber 502 configured to receive a second reagent. The second chamber 502 is connected to the metering chamber 532 via a fluid channel 556 and a passive valve 522. The fluid structure also includes a fluid channel 560 connected to the first chamber 504.

[0194] The sample and a first reagent are introduced into the first chamber 504 to form a sample mixture 1. The sample mixture 1 enters the metering chamber 532 by applying a driving mechanism as described herein. The driving mechanism is removed after the sample mixture 1 contacts the hydrophilic surface 534 of the metering chamber 532. However, as... Figure 5H As shown, driven by capillary force, sample mixture 1 continues to flow in the metering chamber and then stops at capillary valve 528. Figure 5I As shown, after the first portion of sample mixture 1 is collected in metering chamber 532, the remaining portion of sample mixture 1 in first chamber 504 is removed from first chamber 504 via fluid channel 560.

[0195] Then, as Figure 5JAs shown, a first portion of the second reagent in the second chamber 502 and the sample mixture 1 in the metering chamber 532 is introduced into the first chamber 504 via a driving mechanism to form the sample mixture 2. The driving mechanism can be pneumatic force or other types of force. Non-limiting examples of other types of force include, but are not limited to, gravity, capillary force, electrophoresis, magnetism, acoustic pressure, and centrifugal force, and combinations thereof. As a non-limiting example of applying pneumatic force, both vents 512 and 516 are connected to a pneumatic pressure higher than ambient atmospheric pressure, and vent 514 is connected to ambient atmospheric pressure. As another example of applying pneumatic force, both vents 512 and 516 are connected to ambient atmospheric pressure, and vent 514 is connected to a pneumatic pressure lower than ambient atmospheric pressure. In some embodiments, the pneumatic pressure at vents 512 and 516 can be used simultaneously to drive the second reagent and the first portion of the sample mixture 1 into the first chamber 504. In other embodiments, pneumatic pressure may be applied sequentially to vents 512 and 516 to drive a first portion of the second reagent and sample mixture 1 into the first chamber 504.

[0196] Figure 5K Another non-limiting example of a fluid structure for implementing serial dilution is shown, which is used in various sample analysis methods. The fluid structure includes a first chamber 504, a metering chamber 532, and a second chamber 502 connected to the first chamber 504 via a fluid channel 559. The second chamber 502 receives a second reagent prior to serial dilution. After a first portion of sample mixture 1 is collected in the metering chamber 532, the remaining portion of sample mixture 1 in the first chamber 504 is removed from the first chamber 504 via a fluid channel 560. The second reagent and the first portion of sample mixture 1 in the second chamber 502 are then introduced into the first chamber 504 via the driving mechanism described herein to form sample mixture 2.

[0197] In some embodiments, such as Figure 8A As shown, the fluid cartridge 500 is received in the reader 801 for continuous dilution and sample analysis (e.g., cell counting analysis). The reader 801 has a docking port 802 to receive the cartridge into the instrument. In some embodiments, the docking port 802 is covered by a light-shielding door after the cartridge is received, which shields the cartridge from ambient light during sample analysis (e.g., cell counting analysis).

[0198] As a non-restrictive example, Figure 8BThe functional modules of the reader are shown, including a receiving module, an actuation module, a detection module, and an analysis module. The receiving module establishes an interface between the fluid cartridge and the reader. For example, when pneumatic pressure is applied to the vent of the cartridge, a pneumatic connection is required between the vent on the cartridge and the pneumatic source in the reader. The actuation module applies one or more actuation mechanisms to assist in the operation of the cartridge. For example, the actuation module includes a pneumatic source as described herein. Alternatively, the actuation module includes a mechanical actuator to open an active valve in the fluid cartridge. The detection module measures signals from the cartridge for sample analysis (e.g., cell counting analysis). The analysis module processes the measured signals to transmit measurement results (e.g., results of a complete blood count test).

[0199] In some embodiments, after the fluid cartridge is received by the reader, the fluid cartridge is positioned such that gravity pulls the fluid reagent away from the chamber vent and toward the bottom of the chamber. Figure 8C A non-limiting example of this orientation is shown. In this example, gravity pulls the fluid reagent 581 away from the vent 511 and towards the bottom of the chamber 501. In this orientation, a gas gap is formed between the vent 511 and the fluid reagent 581. Simultaneously, the channel 551 enters the chamber 501 at a position lower in height than the fluid sample 581. Thus, the fluid reagent 581 can be driven into the channel 551 to form a diluted sample mixture.

[0200] In various embodiments, the cartridge is positioned such that gravity pulls the fluid reagent away from the chamber vent and toward the bottom of the chamber. In various embodiments, the chamber is configured to store the fluid reagent. In this orientation, a gas gap is formed between the vent and the fluid reagent. Simultaneously, a channel is inserted into the chamber below the upper surface of the fluid reagent. Thus, the fluid reagent can be driven into the channel to form a diluted sample mixture.

[0201] In various embodiments, the cartridge is positioned such that gravity pulls the fluid reagent away from the chamber vent and toward the bottom of the chamber. In various embodiments, the chamber is configured to form a diluted sample mixture. In this orientation, a gas gap is formed between the vent and the diluted sample mixture. Simultaneously, a channel accesses the chamber below the upper surface of the diluted sample mixture. Thus, the diluted sample mixture can be transferred out of the chamber via the channel for further processing. In some embodiments, the diluted sample mixture is transferred out of the chamber via the channel and into a flow chamber to form a sample stream for cell counting analysis. In some embodiments, bubbles are introduced into the sample mixture to aid mixing between the sample and reagent, and gravity helps the bubbles drift toward the vent. As the bubbles drift to the upper surface of the sample mixture and burst, the number of bubbles in the sample mixture decreases.

[0202] Figure 9A This paper illustrates a non-limiting example of measuring red blood cells and platelets in a fluidic chamber using a serial dilution method as described herein. In this example, the first or second reagent comprises a fluorescent dye, and optical signals comprising fluorescence and light scattering are measured to detect red blood cells and platelets. The fluorescence intensity and scattered light intensity of the detected particles are analyzed in the form of a scatter plot, and the detected particles are classified into three distinct clusters corresponding to red blood cells, platelets, and debris.

[0203] Figure 9B A non-limiting example of measuring leukocytes in a fluidic chamber using a serial dilution method as described herein is shown. In this example, the first or third reagent comprises a fluorescent dye, and optical signals comprising fluorescence and light scattering are measured to detect leukocytes. The fluorescence intensity and scattered light intensity of the detected particles are analyzed in the form of a scatter plot, and the detected leukocytes are classified into four distinct clusters corresponding to four leukocyte subtypes, including lymphocytes, monocytes, neutrophils, and eosinophils.

[0204] The serial dilution methods described herein for sample analysis (e.g., cell count analysis) can be used to measure additional whole blood cell count parameters, including but not limited to hemoglobin, hematocrit, reticulocyte count, nucleated red blood cell count, erythrocyte indices (e.g., mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and erythrocyte distribution width), and platelet indices (e.g., mean platelet volume, plateletcrit, platelet distribution width, and platelet-macrocell ratio).

[0205] Numerous variations and alternative elements have been disclosed in the embodiments of this disclosure. Further variations and alternative elements will be apparent to those skilled in the art. These variations include, but are not limited to, the fluid units, components, and structures selected for the apparatus and methods of this disclosure, and the samples that can be analyzed therefrom. Various embodiments of this disclosure may specifically include or exclude any of these variations or elements.

[0206] In some embodiments, the numbers used to describe and claim certain embodiments of this disclosure, representing the amount and properties of components, such as concentration, reaction conditions, etc., should be understood to be modified by the term "about" in some cases. As a non-limiting example, those skilled in the art generally consider a difference (increase or decrease) of no more than 10% in values ​​to mean the term "about". Therefore, in some embodiments, the numerical parameters set forth in the written description and appended claims are approximate values ​​that may vary depending on the desired properties sought from a particular embodiment. In some embodiments, numerical parameters should be interpreted based on the number of significant figures reported and by applying conventional rounding techniques. Although the wide range of numerical values ​​and parameters described in some embodiments of this disclosure are approximate, the values ​​set forth in specific embodiments are reported as accurately as possible. The numerical values ​​presented in some embodiments of this disclosure may contain some errors necessarily caused by the standard deviation found in their respective test measurements.

[0207] The grouping of alternative elements or embodiments disclosed herein should not be construed as limiting. Elements of each group may be referenced and claimed individually or in any combination with other elements of that group or other elements found herein. For convenience and / or patentability reasons, one or more elements of a group may be included in or removed from that group. When any such inclusion or removal occurs, this specification is deemed to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

[0208] This disclosure is explained through various examples, which are intended purely as examples and should not be construed as limiting the disclosure in any way. Various examples are provided to better illustrate the claimed disclosure and should not be construed as limiting the scope of this disclosure. The specific materials mentioned are for illustrative purposes only and are not intended to limit the disclosure. Those skilled in the art can develop equivalent means or reactants without practicing inventive skills and without departing from the scope of this disclosure.

[0209] The various methods and techniques described above provide multiple ways to implement this application. It should be understood, of course, that not all the described objectives or advantages can necessarily be achieved according to any particular embodiment described herein. Therefore, those skilled in the art will recognize, for example, that these methods can be implemented in a manner that achieves or optimizes one or more advantages taught herein, without necessarily achieving other objectives or advantages taught or suggested herein. Various alternatives are mentioned herein. It should be understood that some preferred embodiments specifically include one, another, or several features, while other embodiments specifically exclude one, another, or several features, and other embodiments reduce specific features by including one, another, or several advantageous features.

[0210] Furthermore, those skilled in the art will recognize the applicability of the various features derived from the various embodiments. Similarly, the various elements, features, and steps discussed above, as well as other known equivalents of each such element, feature, or step, can be used by those skilled in the art in various combinations to perform methods based on the principles described herein. Among the various elements, features, and steps, some will be specifically included and others will be specifically excluded in different embodiments.

[0211] Although this application has been disclosed in the context of certain embodiments and examples, those skilled in the art will understand that embodiments of this application extend beyond the specifically disclosed embodiments to other alternative embodiments and / or their uses, modifications, and equivalents.

[0212] This document describes preferred embodiments of the present application, including the best modes known to the inventors for carrying out the present application. Variations of those preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. It is conceivable that those skilled in the art may suitably employ such variations, and that the present application may be implemented in ways other than those specifically described herein. Therefore, many embodiments of the present application include all modifications and equivalents of the subject matter listed in the appended claims as permitted by applicable law. Furthermore, unless otherwise stated herein or clearly contradicted by the context, the present application covers any combination of the foregoing elements in all possible variations.

[0213] All patents, patent applications, publications of patent applications, and other materials cited herein, such as articles, books, specifications, publications, documents, things, and / or the like, except for any history of prosecution documents relating to them, or any history of prosecution documents that is inconsistent with or conflicts with this document, are incorporated herein by reference in their entirety for all purposes. Or any claim that may have a limiting effect on the widest scope of the claims now or hereafter associated with this document. For example, if there is any inconsistency or conflict between the use of terminology in the specification, definitions, and / or related to any introduced material and the use of terminology related to this document, the description, definition, and / or use of terminology in this document shall prevail.

[0214] It should be understood that the embodiments of this application disclosed herein illustrate the principles of the embodiments of this application. Other modifications that may be adopted are within the scope of this application. Therefore, as examples and not limitations, alternative configurations of the embodiments of this application can be utilized based on the teachings herein. Therefore, the embodiments of this application are not limited to the embodiments precisely shown and described.

[0215] Various embodiments of this disclosure have been described above in a detailed description. While these descriptions directly depict the above embodiments, it should be understood that modifications and / or variations to the specific embodiments shown and described herein will occur to those skilled in the art. Many such modifications or variations falling within the scope of this specification are also included therein. Unless specifically indicated, it is intended by the inventors that the words and phrases in this specification and claims be given their common and customary meanings to those skilled in the art.

[0216] The foregoing description of various embodiments of this disclosure known to the applicant at the time of filing this application is intended for illustrative and descriptive purposes. This specification is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed, and many modifications and variations can be made in accordance with the foregoing teachings. The described embodiments are intended to explain the principles of this disclosure and its practical application, and to enable those skilled in the art to utilize this disclosure in various embodiments and with various modifications suitable for the particular intended use. Therefore, it is intended that this disclosure be limited to the specific embodiments disclosed for implementing this disclosure.

[0217] While specific embodiments of this disclosure have been shown and described, it will be apparent to those skilled in the art that changes and modifications may be made based on the teachings herein without departing from this disclosure and its broader aspects, and therefore, the appended claims include all such changes and modifications within their scope, as in the true spirit and scope of this disclosure.

[0218] Other aspects of this disclosure

[0219] The various aspects of the subject matter described herein may be used alone or in combination with any one or more of the other aspects described herein. Without limiting the foregoing description, in a first aspect of this disclosure, an apparatus comprising a fluid cartridge includes: a first chamber configured to receive a sample and a first reagent to form a sample mixture 1; and a metering chamber connected to the first chamber and configured to meter a first portion of the sample mixture 1.

[0220] According to a second aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, wherein the metering chamber includes a surface, and at least a portion of the surface is hydrophilic.

[0221] According to a third aspect of this disclosure, it can be used in combination with any other aspect or combination thereof listed herein, and the metering chamber includes a capillary valve.

[0222] According to the fourth aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, with the metering chamber connected to the vent.

[0223] According to the fifth aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, wherein the metering chamber is connected to a chamber having a vent.

[0224] According to the sixth aspect of this disclosure, it can be used in combination with any other aspect or combination thereof listed herein, and the connection between the metering chamber and the chamber having a vent includes a valve.

[0225] According to the seventh aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, wherein the first reagent is a liquid reagent.

[0226] According to the eighth aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, and the apparatus described herein also includes a reader configured to receive a fluid cartridge and perform sample analysis.

[0227] According to the ninth aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, wherein the reader is configured to apply a driving mechanism to the fluid cartridge to measure a first portion of the sample mixture 1 in the metering chamber.

[0228] According to the tenth aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, and the driving mechanism is aerodynamic.

[0229] According to the eleventh aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein to initiate a drive mechanism to drive the sample mixture 1 to contact a hydrophilic surface in the metering chamber.

[0230] According to the twelfth aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein to stop the drive mechanism when the sample mixture 1 comes into contact with the hydrophilic surface in the metering chamber.

[0231] According to the thirteenth aspect of this disclosure, it can be used in combination with any other aspect or combination thereof listed herein, wherein the reader is configured to receive the fluid cartridge by gravity in a direction that pulls the fluid in the chamber of the fluid cartridge away from the vent of the chamber.

[0232] According to the fourteenth aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, and the fluid cartridge further includes a fluid structure configured to mix a first portion of sample mixture 1 and a second reagent to form sample mixture 2.

[0233] According to the fifteenth aspect of this disclosure, which can be used in combination with any other aspect or combination of aspects listed herein, the fluid structure includes a second chamber connected to the metering chamber, and the second chamber is configured to receive the second reagent prior to the first portion of the second reagent and sample mixture 1 being mixed.

[0234] According to the sixteenth aspect of this disclosure, it can be used in combination with any other aspect or combination thereof listed herein, and the connection between the metering chamber and the second chamber includes a valve, a passive valve, or an active valve.

[0235] According to the seventeenth aspect of this disclosure, it can be used in combination with any other aspect or combination thereof listed herein, and the fluid cartridge also includes a reagent storage unit comprising a storage chamber configured to contain fluid and a valve with a breakable connection.

[0236] According to the eighteenth aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, and the fluid cartridge further includes a flow chamber configured to form a sample flow from the sample mixture in the fluid cartridge.

[0237] According to the nineteenth aspect of this disclosure, which can be used in combination with any other aspect or combination thereof listed herein, a method comprising: receiving a sample using a fluid cartridge, wherein the fluid cartridge includes: a first chamber configured to receive the sample and a first reagent to form a sample mixture 1; a metering chamber connected to the first chamber and configured to meter a first portion of the sample mixture 1; and placing the fluid cartridge into a reader for sample analysis of the sample.

[0238] According to the twentieth aspect of this disclosure, which can be used in combination with any other aspect or combination thereof listed herein, the reader applies a driving mechanism to the fluid cartridge to measure a first portion of the sample mixture 1 in the metering chamber.

[0239] According to the twenty-first aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, after the first portion of sample mixture 1 is metered, sample mixture 1 is removed from the first chamber.

[0240] According to the twenty-second aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, wherein the fluid cartridge is configured to mix a second portion of sample mixture 1 with a third reagent to form sample mixture 3.

[0241] According to aspect twenty-three of this disclosure, it may be used in combination with any other aspect or combination of aspects listed herein, wherein the third reagent is a dried or dehydrated reagent.

[0242] According to the twenty-fourth aspect of this disclosure, it can be used in combination with any other aspect or combination thereof listed herein, the fluid cartridge further includes a flow chamber configured to form a sample stream from a sample mixture in the fluid cartridge, and wherein a reader measures cells, particles, or analytes, or combinations thereof, in the sample stream.

[0243] According to the twenty-fifth aspect of this disclosure, which can be used in combination with any other aspect or combination thereof listed herein, the fluid cartridge further includes two reagent storage units, and each reagent storage unit includes a storage chamber configured to contain fluid and a valve with a breakable connection.

[0244] According to the twenty-sixth aspect of this disclosure, which can be used in combination with any other aspect or combination thereof listed herein, a method comprising: receiving a sample and a first reagent using a fluid cartridge to form a sample mixture 1; measuring a first portion of the sample mixture 1 in the fluid cartridge using a metrology chamber; mixing the first portion of the sample mixture 1 with a second reagent using the fluid cartridge to form a sample mixture 2; and measuring a signal from the sample mixture 1, or the sample mixture 2, or both, in the fluid cartridge using a reader.

[0245] According to the twenty-seventh aspect of this disclosure, it can be used in combination with any other aspect or combination thereof listed herein, wherein the reader applies a driving mechanism to the metering chamber to meter a first portion of the sample mixture 1.

[0246] According to aspect twenty-eight of this disclosure, it can be used in combination with any other aspect or combination thereof listed herein, wherein the molar osmotic concentration of the first reagent is about 140-160 mOsm / L, 160-180 mOsm / L, 180-200 mOsm / L, 200-220 mOsm / L, 220-240 mOsm / L, 240-260 mOsm / L, 260-280 mOsm / L, 280-300 mOsm / L, 300-320 mOsm / L, 320-340 mOsm / L, 340-360 mOsm / L, 360-380 mOsm / L, or 380-400 mOsm / L.

[0247] According to the twenty-ninth aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, wherein a red blood cell lysis compound is introduced into a chamber, and then a first portion of sample mixture 1 and a second reagent are introduced into the chamber to form sample mixture 2.

[0248] According to the thirtieth aspect of this disclosure, it may be used in combination with any other aspect or combination thereof listed herein, such as the method herein further comprising using a flow chamber in a fluid cartridge to form a sample stream from a sample mixture in the fluid cartridge, and using a reader to measure cells, particles, or analytes, or combinations thereof, in the sample stream.

[0249] According to the thirty-first aspect of this disclosure, it may be used in combination with any other aspect or combination thereof listed herein, such as the method herein further comprising using a reader to measure cells, red blood cells, platelets, particles, or analytes, or combinations thereof, in a sample stream formed from sample mixture 2.

[0250] According to the thirty-second aspect of this disclosure, it can be used in combination with any other aspect or combination thereof listed herein, such as the method described herein further comprising using a fluid cartridge to mix a second portion of sample mixture 1 and a third reagent to form sample mixture 3.

[0251] According to aspect thirty-three of this disclosure, it may be used in combination with any other aspect or combination thereof listed herein, the third reagent including erythrocyte lysis compounds.

[0252] According to the thirty-fourth aspect of this disclosure, it can be used in combination with any other aspect or combination of aspects listed herein, such as the method herein further comprising using a flow chamber in a fluid cartridge to form a sample stream from the sample mixture 3 in the fluid cartridge, and using a reader to measure cells, leukocytes, hemoglobin, particles, or analytes, or combinations thereof, in the sample stream formed from the sample mixture 3.

[0253] According to the thirty-fifth aspect of this disclosure, it may be used in combination with any other aspect or combination thereof listed herein, such as the method herein further comprising measuring sample mixture 2 in the flow chamber of the fluid cell prior to measuring sample mixture 3 in the flow chamber of the fluid cell.

Claims

1. An apparatus for continuously diluting a sample mixture, characterized in that, The device includes a fluid cartridge, the fluid cartridge comprising: A first chamber, the first chamber being configured to receive a sample and a first reagent to form a first sample mixture; A metering chamber configured to meter a first portion of the first sample mixture; A third chamber, the third chamber being configured to receive a second portion of the first sample mixture with a third reagent to form a third sample mixture; The metering chamber is connected to the first chamber, and the third reagent is a dried or dehydrated reagent. The metering chamber has a hydrophilic surface that introduces capillary force to pull the first sample mixture. The fluid cartridge also includes a driving mechanism configured to drive the first sample mixture to contact the hydrophilic surface of the metering chamber. After the first sample mixture contacts the hydrophilic surface of the metering chamber, the driving mechanism is stopped, and the first sample mixture driven by the capillary force continues to flow and fill the metering chamber.

2. The apparatus according to claim 1, characterized in that, The driving mechanism is pneumatic.

3. The apparatus according to claim 1, characterized in that, The fluid cartridge further includes a second chamber configured to receive a first portion of the first sample mixture with a second reagent to form a second sample mixture.

4. The apparatus according to any one of claims 1 to 3, characterized in that, The fluid cartridge also includes a flow chamber configured to form a sample flow from a sample mixture in the fluid cartridge, the sample mixture being any sample mixture in the fluid cartridge.

5. The apparatus according to claim 4, characterized in that, The flow chamber is a sheathless flow chamber, through which the sample stream flows without a sheath, and the sample stream has a diameter equal to that of the flow chamber.

6. A method for continuously diluting a sample mixture, comprising a fluid cartridge including a first chamber, a metering chamber, and a third chamber, characterized in that... The method includes: receiving a sample and a first reagent in a first chamber to form a first sample mixture; measuring a first portion of the first sample mixture in a metering chamber; and receiving a second portion of the first sample mixture and a third reagent in a third chamber to form a third sample mixture; wherein the metering chamber and the third chamber are respectively connected to the first chamber, and the third reagent is a dried or dehydrated reagent; the metering chamber has a hydrophilic surface that introduces capillary forces to pull the first sample mixture; the fluid cartridge further includes a driving mechanism configured to drive the first sample mixture to contact the hydrophilic surface of the metering chamber, and after the first sample mixture contacts the hydrophilic surface of the metering chamber, stopping the driving mechanism, and allowing the first sample mixture driven by the capillary forces to continue flowing and filling the metering chamber.

7. The method according to claim 6, characterized in that, The method further includes: using a second chamber to receive a first portion of the first sample mixture with a second reagent to form a second sample mixture.

8. The method according to claim 6, characterized in that, The method further includes: using a reader to measure at least one substance in the sample stream formed from the sample mixture, including cells, particles, or analytes.

9. The method according to claim 6, characterized in that, The third reagent includes a red blood cell lysis compound.

10. The method according to claim 6, characterized in that, The fluid cartridge further includes a flow chamber, and the method further includes: using the flow chamber to form a sample stream from a sample mixture in the fluid cartridge, and using a reader to measure at least one substance from the sample stream formed from the sample mixture, namely cells, hemoglobin, particles, or analytes.

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