Automated microscopic cell analysis

Abstract

To provide single-use test cartridges, cell analyzer apparatus, and methods for automatically performing microscopic cell analysis tasks, such as counting and analyzing blood cells in biological samples.SOLUTION: In one general aspect, a small measured quantity of a biological sample, such as whole blood, is placed in a mixing bowl on the disposable test cartridge and then is inserted into the cell analyzer. The analyzer also deposits a known amount of diluent / stain in the mixing bowl and mixes the diluent / stain with the blood. The analyzer takes a measured amount of the mixture and dispenses in a sample cup on the cartridge in fluid communication with an imaging chamber. Geometry of the imaging chamber is chosen to maintain uniformity of the mixture, and to prevent cells from crowding or clumping as it is transferred into the imaging chamber by the analyzer. Images of all of the cellular components within the imaging chamber are counted and analyzed to obtain a complete blood count.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to U.S. Application No. 11 / 616,327, filed June 7, 2017, U.S. Application No. 15 / 221,285, filed July 27, 2016, U.S. Application No. 15 / 017,498, filed February 5, 2016, U.S. Application No. 14 / 947,971, filed November 20, 2015, U.S. Provisional Application No. 62 / 138,359, filed March 25, 2015, U.S. Provisional Application No. 62 / 113,360, filed February 6, 2015, and U.S. Provisional Application No. 62 / 084,760, filed November 26, 2014. This application also claims priority to U.S. Provisional Application No. 62 / 360,236, filed July 8, 2016, and U.S. Provisional Application No. 62 / 394,702, filed September 14, 2016. All of the above-listed applications are incorporated herein by reference.

[0002] The present invention relates to an analyzer and method for automatically performing microscopic blood cell analysis tasks, such as counting blood cells in a biological sample. More particularly, the present disclosure relates to single-use devices, apparatus, and methods used to count red blood cells, white blood cells, and platelets, and measurements related to these particles. [Background technology]

[0003] There are many different methods for enumerating particles such as blood cells in a biological sample. Determining the number of blood cells per unit volume in a sample provides physicians with important diagnostic information. The most basic method of counting blood cells consists of introducing a diluted biological sample into a hemocytometer and examining it using a microscope. A hemocytometer is a device with an optically transparent chamber of known depth, typically 100 microns deep, and with graduated markings that define unit volumes, typically .01 μL. For example, a homogenous mixture of diluted whole blood can be introduced into the hemocytometer by capillary action to form a monolayer. Using a microscope to visualize the diluted sample, different types of blood cells can be manually counted within a limited number of marked ranges. The counts can be summed to calculate the number of blood cells per unit volume. This manual method is laborious, time-consuming, requires a skilled technician to operate the microscope and recognize the various types of blood cells, and is prone to error. Its accuracy is limited by the number of blood cells being counted and the uniformity of the monolayer formed by the introduction of the diluted sample.

[0004] As a result, automated methods such as impedance analysis (Coulter principle, U.S. Pat. No. 2,656,508) and flow cytometry have been developed to rapidly count, size, and classify relatively large numbers of blood cells for diagnostic tests such as a complete blood count (CBC) with five part differential, sometimes called a CBC. These automated methods also have drawbacks. The analyzers are relatively large and expensive, and require skilled operators to use and maintain them. Such analyzers are generally only available in centralized laboratories. Blood samples are collected in special containers with anticoagulants to prevent the blood from clotting during transport to the laboratory. This process adds costs and risks of erroneous results due to transport, handling, labeling, and transcription, as well as time delays in obtaining results. These analyzers also flag or reject more than 20% of the samples tested for further investigation by manual differentiation. Only highly skilled technicians can perform manual differentiation. Flags are generally generated by impedance or flow cytometry counters due to ambiguities in the impedance or diffusion profiles of populations of blood cells. Microscopic imaging analysis is used as a reference method since it does not suffer from the same ambiguities as impedance methods and flow cytometry. Similarly, automated imaging analysis has a much lower flag rate.

[0005] CBC generally includes measurements of white blood cells (WBC) per unit volume, red blood cells (RBC) per unit volume, platelets (PLT) per unit volume, hematocrit (HCT) or packed cell volume (PCV), hemoglobin (HGB), and measurements on red blood cells including mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin content (MCHC), and red blood cell distribution width (RDW). Diagnostic tests sometimes referred to as "CBC with differential" or "CBC with 5-way differential" further include neutrophil granulocytes (NEU), lymphocytes (LYM), monocytes (MON), eosinophils (EO), and basophils (BASO) per unit volume or as a percentage of white blood cells (WBC). A CBC with differential may also include immature cells (IC), atypical lymphocytes, nucleated red blood cells (nRBC), and reticulocytes (RETIC) per unit volume of blood sample.

[0006] CBC provides a panel of blood cell measurements that can be used to diagnose a wide variety of abnormal conditions, such as anemia or infection, or to monitor a patient's treatment, such as chemotherapy. Due to its availability, CBC analysis is one of the most commonly performed diagnostic tests in medicine, but patients typically wait a day or several days to get the results. If microscopic blood cell analysis could be performed on a portable, easy-to-use analyzer closer to the patient, the results could be more directly translated into improved patient care. A simple system that could provide a CBC at the clinic or bedside, in a specialized critical care unit (CCU) or intensive care unit (ICU), or in a hospital emergency room, using a drop of blood from a fingertip within minutes, could have a tremendous impact on health care delivery and availability.

[0007] Recent patent documents describe devices that perform blood cell analysis of blood samples that are simpler than centralized laboratory hematology analyzers. In U.S. Patent No. 7,771,658 issued to Larsen, the applicant provides a technique for performing flow cell analysis of blood cells in a single-use disposable cartridge. Larsen describes a means for taking a precise amount of blood sample, diluting the blood amount with a precise volume of diluent, and mixing the blood with the diluent to obtain a homogenous solution. Larsen utilizes a single-use cartridge to flow a measured amount of the sample and diluent mixture through an orifice at a rate of several thousand particles per second, where the particles are counted, sized and classified for analysis according to the Coulter Principle. Because Larsen's disclosure is directed to the analysis of small samples of whole blood, errors in the metering of various volumes, or in the mixing or sampling steps, can significantly affect the accuracy of the results.

[0008] PCT Patent Application Publication WO2014099629 issued to Ozcan et al. describes a system for analyzing blood samples using a mobile electronic device with a camera. The sample preparation process for each test requires precise measurement of 10 μL of whole blood and mixing with 85 μL of phosphate buffered saline and 5 μL of a nucleic acid stain. 10 μL of this diluted mixed sample is then loaded into a blood cell counting chamber with a precise channel height of 100 μm and imaged by a digital camera. Separate blood cell counting chambers are required for the analysis of red blood cells, white blood cells, and hemoglobin, and each test must be performed separately. The accuracy of the final result depends on precise measurement of the various sample preparation steps, including precise metering of sample and diluent, as well as precise machining of the counting chamber. Uniform and consistent maintenance of the chamber height dimension of 100 μm in a disposable cartridge is difficult to achieve in a low-cost device.

[0009] Wang et al., U.S. Patent No. 8,837,803, describes a method for determining the corpuscular volume of red blood cells that aims to avoid errors associated with dilution, mixing, and sampling by analyzing a sample of substantially undiluted whole blood. Although this approach is attractive, working with undiluted whole blood is difficult. Blood cells are so numerous (e.g., 5,000,000 red blood cells in 1 μL) that their distribution can be affected by contact with the surfaces of the disposable cartridge. In addition, to image blood cells in this crowded environment, the imaging chamber must be only a few microns deep to prevent the cells from overlapping, and must be precisely machined, as this determines the volume of the blood sample to be analyzed.

[0010] Therefore, a compact and precise method of performing microscopic blood cell analysis using digital camera imaging of diluted samples that does not require precise measurement of diluent volume or precise or exact dimensions of the imaging chamber is highly desirable. Summary of the Invention

[0011] It is an object of the present invention to provide an apparatus that can provide a count of blood cells or other particles of a biological sample per unit volume without requiring an operator with specialized knowledge or skills. Another object of the present invention is to provide an easy to use analyzer that can perform a full CBC measurement in a few minutes. Another object of the present invention is to provide an analyzer that performs a CBC on a small fingerstick sample. Another object of the present invention is to provide a method for determining the concentration of particles in a diluted biological sample that does not require precise measurement of the volume of diluent or reagent to be mixed with the sample. Another object of the present invention can provide ready-to-use reagents and diluents that are used to perform microscopic analysis of a biological sample that does not require preparation, mixing, or measurement by the user. Another object of the present invention is to create a substantially homogenous monolayer of blood cells of a diluted sample in a single-use disposable device and perform a CBC analysis in a few minutes. Another object is to provide a sample collection device that can be easily operated by a user to obtain a biological sample for analysis. Yet another object is to provide all the fluidic components of a CBC analyzer in a single-use device to prevent cross-contamination between samples and to eliminate the need to clean the fluidic components of the analyzer. Another object of the present invention is to provide internal monitoring of the critical process step of counting blood cells so that potential erroneous results can be flagged. Yet another object is to provide a method of performing a CBC on a sample immediately after collection, eliminating transport of the sample to a laboratory or storage of the sample. Any or all of these and / or other objects or advantages may be accomplished by embodiments of the present invention as provided in the appended claims.

[0012] It is a further object of the present invention to provide a hematology analyzer that performs CBC using imaging techniques instead of the Coulter principle of flow cytometry. Another object of the present invention is to provide a hematology analyzer that utilizes a single-use disposable imaging chamber. Another object of the present invention is to provide a substantially homogenous monolayer of blood cells and platelets in unrestrained diluted whole blood as opposed to mechanically deformed, distorted or flattened blood cells smeared or sprayed onto a glass slide, or blood cells sprayed or deposited and fixed onto a glass slide. Another object of the present invention is to provide an imaging-based hematology analyzer that flags samples at a lower rate than hematology analyzers that use impedance or flow cytometry.

[0013] The present disclosure provides improved methods, systems, and devices for performing particle counting and measurement in biological samples. Aspects of the present disclosure aim to determine the concentration of one or more types of particles in a biological sample and provide results as a number of such particles per unit volume within minutes. Particles may be any mass suspended in a liquid that can be recognized by optical inspection using automated microscopy and image analysis techniques well known in the art. Examples of particles include blood cells, platelets, sperm, bacteria, spores, and These include, but are not limited to, inorganic particles.

[0014] Although embodiments of the present invention can be used in many applications for counting particles suspended in a liquid, the present disclosure demonstrates the benefit of the present invention in performing a complete blood count (CBC) or CBC with differential on a human or animal blood sample, as defined above. The present disclosure describes a single-use test cartridge for use with an apparatus, including an automated microscope, for analyzing blood cells in a biological sample. The test cartridge is used to collect the biological sample. For example, a user can prick a patient's finger, obtain a whole blood sample, and collect the resulting blood droplet in the test cartridge. The user can accomplish this by holding the test cartridge under a drop of liquid dripping from the patient's finger and then moving the drop closer until it touches an input port or sample cup of the test cartridge. In an alternative embodiment, a user may take a blood sample in a vein and transfer it to the test cartridge using a plastic bulb pipette. In one example, the single-use test cartridge includes an input port adapted to accept a variety of biological sample sources, including a direct sample, a capillary tube, or a transfer pipette. In another example, the test cartridge can draw the sample in by capillary action. The test cartridge may also include a closure that the user applies to cover the input port after the sample is collected in the test cartridge. The closure may be part of the test cartridge or a separate device. The user may manually apply the closure or the instrument may automatically move the closure to cover the input port when the test cartridge is docked to the instrument. The closure provides a physical barrier to avoid contact with excess sample that is subsequently on the surface of the test cartridge. The closure has a vent that provides an air path to the input port so that the liquid sample can move within the test cartridge when a vacuum is applied, as described below.

[0015] Within the test cartridge, the input port is connected by an input channel to a metering chamber that can accurately separate small volumes of sample from unmeasured samples. As an example, the collected sample volume may be 5-20 μL, of which the metering chamber separates or isolates 0.1-5 μL for measurement. If fingerstick samples are utilized, a sample volume of less than 10 μL minimizes the risk of hemolysis and dilution by interstitial fluid. The metering chamber can be a channel, cavity, or through conduit in a valve, or a compartment of another three-dimensional shape that can be reproducibly fabricated to contain a predetermined volume of biological sample in the range of 0.1-5 μL. Injection molding, compression molding, etching, or other processes known to those skilled in the art of creating lab-on-a-chip or microfluidic devices can be utilized to fabricate the metering chamber.

[0016] In one example, the metering chamber is combined with the rotary valve structure by molding the through conduit on the cylindrical stem of the rotary valve. As an example, a through conduit with a cylindrical cross section of 0.5 mm diameter and 5 mm length has an internal volume of about 1 μL. The through conduit is first filled with a biological sample by providing a fluid communication path between the input port and the input channel, the through conduit, and the vacuum channel. The sample is drawn into the through conduit by a vacuum applied to the vacuum channel. Alternatively, the biological sample can be drawn into the through conduit by capillary action. Once the biological sample is completely filled into the through conduit, the cylindrical stem of the rotary valve rotates until the through conduit is no longer connected to the input channel or the vacuum channel, thereby separating or isolating the sample volume contained in the through conduit. Alternatively, a rotating face seal valve, a slide valve, or a fixed volume in the test cartridge can be used to isolate a small amount of sample.

[0017] Additionally, the test cartridge elements may include chambers containing or storing prepackaged liquid reagents, mixing chambers, imaging chambers, and sample and The metering chamber includes a through conduit, along with a fluid channel through which the liquid reagent may travel. In one example, after the biological sample fills the through conduit, the rotary valve is positioned such that the through conduit is placed in fluid communication with both the liquid reagent and the mixing chamber. Empirical studies have determined that a volume of liquid reagent or diluent or stain (hereinafter "diluent / reagent") greater than three times the volume of the through conduit is sufficient to flush the entire isolated sample. In one example, the diluent / reagent is a combination of diluent and stain, and is provided in a volume that results in a dilution ratio of approximately 40:1. Thus, 40 times the volume of the metering chamber can be used to push the isolated sample out of the through conduit and into the mixing chamber, where the sample is uniformly mixed with the diluent / reagent, and then transferred to the imaging chamber.

[0018] The single-use test cartridge contains an imaging chamber through which an automated microscope can acquire images of blood cells in a mixture of diluent / reagent and sample. In one embodiment of the present invention, the isolated sample volume is diluted with a known amount of diluent / reagent, thereby establishing a dilution ratio. The amount of diluent / reagent may be determined by measuring it, for example, by monitoring its flow in a fluidic channel of known dimensions with a camera or other fluid sensor such as optical reflection / transmission or ultrasound, or by weighing what enters a chamber of known volume temporarily and then using only that known volume for sample dilution. According to this embodiment, blood cells in a known volume of the diluted sample are counted and the dilution ratio is known, so that the blood cell count per unit volume of blood sample can be determined. The volume of the diluted sample can be determined by filling an imaging chamber of known volume with the diluted sample. The volume of the imaging chamber may be known by using a highly reproducible manufacturing process or by measuring each test cartridge during manufacturing and encoding sizing parameters into the labeling of the package. Alternatively, a measured amount of the diluted sample can be transferred to an imaging chamber of unknown volume and all blood cells in the chamber are counted. The measured amount of the diluted sample can be determined by monitoring its flow from the mixing chamber into a fluidic channel of known dimensions with a camera and isolating a segment. The segment is then moved into the imaging chamber.

[0019] In a preferred embodiment, the isolated sample volume is transferred to a mixing chamber and then to an imaging chamber. According to this embodiment, neither the dilution ratio nor the volume of the diluent need to be known. The size of the imaging chamber is chosen to ensure that the entire volume of the isolated sample and diluent / reagent can be accommodated within the imaging chamber, but the exact dimensions or volume of the chamber need not be known. The depth of the imaging chamber must be shallow enough to prevent overlapping of the blood cells at the selected dilution ratio when they settle to the bottom. This depth is preferably about 10 μm to 200 μm. In one embodiment, the imaging chamber has a depth of 100 μm and a ratio of diluent / reagent to isolated sample is 40 to 1. The width of the imaging chamber is chosen to provide a smooth flow with uniform filling by the different blood cell sizes without void formation or crowding of the blood cells. The length of the imaging chamber is calculated based on the depth and width parameters to provide the volume required to receive the entire isolated sample at the selected dilution ratio and provide a safety margin. The shape of the imaging chamber may also be chosen to match the field of view of a digital camera, or to facilitate the acquisition of multiple images, or to maintain a uniform distribution of blood cells.

[0020] The inventors have found that if the imaging chamber is shaped like a square or a rectangle with a length to width ratio of 2:1, the blood cells will not fill the imaging chamber uniformly. If the mixture of sample and diluent / reagent is uniform when it enters the imaging chamber, the blood cells will tend to concentrate and clump together, especially near the sides or edges, and when they settle, the layer at the bottom of the imaging chamber will not be homogenous. It is important to have a substantially homogenous monolayer of blood cells in the imaging chamber, as this will facilitate the counting of all blood cells. ... It has been found that blood cells remain substantially uniformly distributed when the width and depth of the chamber are short compared to the length. Desirably, the length to width ratio of the imaging chamber is greater than 10:1. In various embodiments of the present invention, the shape of the imaging chamber may be serpentine, spiral, or curved like a battlement, depending on the form factor of the test cartridge. In all these cases, the width and depth are short compared to the length, so that blood cells do not clump at the sides or corners of the imaging chamber, and the layer of blood cells that settles to the bottom of the imaging chamber is substantially homogenous. Those skilled in the art will recognize that other geometric shapes of the imaging chamber may also be utilized that maintain the distribution of blood cells when the sample and diluent / reagent mixture is transferred to the imaging chamber.

[0021] The design goal of the imaging chamber is to accommodate all of the cells and diluent / reagent from the original metering chamber in a uniform manner and without significant overlap of the cells as they settle to the bottom of the imaging chamber. The dilution ratio, in combination with the depth of the imaging chamber, can be chosen to minimize overlap. Illustratively, the imaging chamber may have a width of .5 mm to 2.5 mm, a depth of 10 to 200 μm, and a dilution ratio of 10:1 to 100:1.

[0022] Materials in contact with the blood cells outside the imaging chamber may be chosen to have surface properties that minimize adhesion of blood cells. The liquid reagent may contain a surfactant or blood cell sphering agent to facilitate blood cell analysis and may advantageously minimize red blood cells from being lost during transfer or overlapping in the imaging chamber. The volume and flow rate of the liquid reagent may also be chosen to improve the likelihood of transferring all blood cells from the metering or mixing chamber to the imaging chamber and ensuring that the distribution of blood cells remains uniform. Yeh-Chan Ahn describes the complex convection-diffusion phenomenon created in serpentine microchannels with varying curvatures (Investigation of laminar dispersion with optical coherence tomography and optical Doppler tomography, Yeh-Chan Ahn, Woonggyu (See Jung, Jun Zhang, and Zhongping Chen, OPTICS EXPRESS 8164, Vol. 13, No. 20, Oct. 3, 2005). Particles, or blood cells in the present case, in suspension and moving through such microchannels tend to separate according to their size, density, channel geometry, and flow rate. Secondary flows from serpentine geometries can create vortices as the channel curves, remixing the blood cells into the center of the flow at a certain filling rate. This remixing helps maintain a near-uniform distribution of blood cells as the fluid fills the microchannel. In one embodiment, the inventors have found that a serpentine path with a width of 1.25 mm, a turning inner diameter of 1.25 mm, a turning outer diameter of 2.5 mm, a depth of 0.125 mm, a length of 500 mm, and an effective filling rate of about 2 μL / sec ensures that the flow of blood cells remains uniform.

[0023] It should be noted that the time to count all blood cells is directly related to the volume of the metering chamber, which creates a trade-off in overall performance. Fabricating a highly reproducible metering chamber is difficult for very small volumes. However, while it may be easier and more reproducible to fabricate a metering chamber with a volume of 5 μL than one with a volume of 1 μL, counting all blood cells in a 5 μL sample volume and analyzing them may take five times as long as a 1 μL sample volume. An embodiment of the present invention may provide a solution to this challenge. By ensuring that the sample and diluent / reagent are well mixed and that an appropriate dilution ratio is chosen, and by utilizing a serpentine imaging chamber whose length is long compared to its width and depth, the pattern of blood cells across the imaging chamber is relatively uniform and reproducible. Under these conditions, the layer of blood cells that settles to the bottom of the imaging chamber is a substantially homogenous monolayer. As a result, instead of imaging the entire layer and counting all blood cells, a representative image or frame can be obtained that is statistically derived to precisely account for all blood cells. Therefore, the final result can be determined within a certain margin of error, either by actual images or by statistical representation. All blood cells are included in the analysis, regardless of how they are viewed. Illustratively, the camera may take up to 20,000 images at 20x to scan the entire imaging chamber. Alternatively, a statistical representation can be obtained every 10 frames. Another option is to divide the imaging chamber into segments and count blood cells in every other or every second, etc. segment. Another alternative to reduce imaging time would be to scan the entire imaging chamber at 10x / 0.25 numerical aperture (NA) or 4x / 0.1NA to obtain a total WBC and RBC count, and then take images at higher magnification (20X .04NA or higher) to obtain the higher resolution details required to count platelets, reticulocytes, and perform WBC differentials.

[0024] The embodiments of the present invention can achieve precise results independent of many factors that affected the accuracy of prior art results. For example, the diluent / reagent volume and dilution ratio do not affect the results as long as all blood cells from the metered sample volume are transferred to the imaging chamber, do not overlap or cluster, and are presented for analysis. Similarly, the representativeness of the selected portion of the sample and reagent mixture, and the homogeneity of that portion, directly affect the accuracy of the results of prior art methods, but should not affect the embodiments of the present invention. More importantly, the depth and uniformity of the imaging chamber, which were difficult or expensive to control in other prior art attempts, should not affect the accuracy of the results of the embodiments of the present invention.

[0025] The present disclosure further describes a compact and easy-to-use blood cell analyzer. The blood cell analyzer accepts a test cartridge and performs all steps of blood cell analysis without further input from a user. In one embodiment, the test cartridge contains all diluents and reagents necessary to perform a CBC analysis of a patient sample placed therein. In this embodiment, the blood cell analyzer has fluid handling components including positive and negative pressure sources that interface with the test cartridge when it is placed in the analyzer. One or more connections are made between the analyzer and the cartridge to fluidly communicate these pressure sources with channels in the cartridge to provide a transport force to move liquids in the cartridge. The blood cell analyzer also has a mechanical valve driver that operates valves in the test cartridge that control the movement of fluids in the test cartridge. When the test cartridge is placed in the analyzer, the mechanical valve driver is connected to a valve indexer to provide a means to operate the valves in the test cartridge. The blood cell analyzer includes a mechanism to release diluents / reagents stored on board the test cartridge. The blood cell analyzer further includes fluid control logic that automatically controls fluid movement within the test cartridge by activating positive and negative pressure or displacement sources and operating mechanical valve drivers according to preprogrammed sequences. The blood cell analyzer may also include a process monitoring camera positioned to capture digital images of fluid movement within the cartridge. Information from the process monitoring camera can be used to provide feedback on the fluid control logic or on monitoring critical steps.

[0026] The present disclosure also provides devices and methods for managing and / or monitoring the sample collection and preparation process to ensure accurate results. In applications where the present invention is used to perform CBC analysis, if too much time passes between obtaining a blood sample and completing dilution and staining of the sample in the test cartridge, the blood may clot or blood cells may precipitate, resulting in erroneous results. In one embodiment of the present invention that mitigates this risk, the test cartridge is placed on the analyzer in a pull-out tray, which initiates process monitoring within the analyzer. The blood sample is added to the cartridge by dispensing a free-flowing drop, or by using a capillary tube inserted into the test cartridge, or by transferring the sample and placing it with a pipette. Once the sample is added to the cartridge, the cartridge is placed on the analyzer in a pull-out tray, which initiates process monitoring within the analyzer. The blood sample is added to the cartridge by dispensing a free-flowing drop, or by using a capillary tube inserted into the test cartridge, or by transferring the sample and placing it with a pipette. Immediately after the cartridge is loaded, the user presses "Run Sample" to begin the test sequence. Because the cartridge is controlled by the analyzer, the time to collect the sample is known and can be short enough to avoid the need for anticoagulant coating or mixing of the blood sample.

[0027] In an alternative embodiment where the sample can be collected several minutes before processing, an anticoagulant such as K2 or K3 EDTA is provided. This may be accomplished by coating the sample input cup with the anticoagulant, by using anticoagulant-coated capillaries for fingerstick samples, or by sampling from an evacuated blood collection tube such as a Becton Dickenson Vacutainer® that contains the anticoagulant. To control for sedimentation of blood cells, the test cartridge according to this embodiment has a timing indicator that is activated when the input port is opened or when the blood sample is introduced. The timing indicator can be a color-changing chemical reaction, a time-delayed thermal reaction, an analog or digital timer, or other means known in the art. When the user loads the test cartridge into the analyzer, the timing indicator is read and the sample is mixed, if necessary, before proceeding. If too much time has passed, the sample can be rejected to avoid producing erroneous results.

[0028] In yet another embodiment that facilitates remote sample collection, the test cartridge is docked to a carrier system. The carrier system is a handheld or portable device used to facilitate some or all of the sample preparation steps. After blood is added to the test cartridge using any of the methods described above, the carrier system according to this embodiment draws the blood into the cartridge, weighs the sample, performs a quality check, completes the sample preparation, and presents the blood cells for image analysis. The carrier system then ejects the test cartridge for a user to transfer to an imaging analyzer, or the carrier system can be docked to an imaging analyzer for automatic handoff. In this embodiment, anticoagulant coating is not required, and there is no risk of blood cell sedimentation, since the critical steps begin once the sample is placed in the test cartridge.

[0029] Combinations of workflow devices and methods are also contemplated in this disclosure. As an example, a simple carrier system may incorporate a digital timer and a mechanism that can weigh the sample, but does not perform quality checks or complete sample preparation. These additional steps would be performed by a hematology analyzer.

[0030] The blood cell analyzer includes an automated microscope that includes an objective lens, a focusing mechanism, a bright field and / or fluorescent light source or both, a filter, a dichroic mirror, and a digital camera. In some embodiments, the blood cell analyzer may further include an illumination source and a photometric detector that measures light transmission at one or more wavelengths to measure the concentration of an analyte in the sample. For example, the test cartridge may include a photometric chamber in fluid communication with an input port by a fluidic channel, through which a sample may be transferred for photometric analysis, such as hemoglobin measurement.

[0031] As an example, to measure hemoglobin, a blood cell analyzer may have an illumination source consisting of two light emitting diodes (LEDs) excited at wavelengths of 502 nm and 880 nm. The light path of the LEDs and the light intensity detector is approximately 0.08 cm (approximately 0.030 inches) in diameter. The 502 nm LED may be selected for absorbance measurements due to the isosbestic points of oxyhemoglobin (OHb) and deoxyhemoglobin (RHb). Also, at 502 nm, the slopes of the OHb and RHb curves are very shallow, so changes due to variations in oxygen saturation (sO2) levels are minimal. Additionally, the carboxyhemoglobin (COHb) curve is also close to this isosbestic point. Therefore, the contribution from COHb is very small. The 880 nm LED can measure background scattering effects from RBCs, WBCs, lipids, etc. This is especially important when making measurements on whole blood. This measurement may also be performed on hemolysis with or without a reagent to convert hemoglobin to a single form such as reduced hemoglobin, methemoglobin, azide methemoglobin, or cyanomethemoglobin. When converted to a single form of hemoglobin, a different peak wavelength may be used for measuring absorption, such as 540 nm or 555 nm.

[0032] Disclosed and contemplated embodiments also include examples of test cartridges that are not preloaded with reagents, but instead are coupled to a reagent supply module housed on board the hematology analyzer. A user loads the reagent supply module into the hematology analyzer where the module is utilized for multiple test cartridges. When the reagent supply module is depleted or expired, the hematology analyzer alerts the user and will not perform any further tests until the reagent supply module is replaced. The reagent supply module includes a cradle that receives the test cartridge, a container that holds liquid diluent / reagent, a diluent delivery pump in fluid communication with the container, and a diluent / reagent output port constructed to interface with the test cartridge when the cartridge is in the cradle. In one example, the size of the container is of sufficient capacity to provide diluent / reagent to dilute multiple samples (50-100) at a reagent-to-sample ratio of 10:1 to about 250:1. The diluent / reagent supply module may include a self-priming mechanism that primes the liquid reagent and eliminates air bubbles. The reagent supply module may further include a chamber to collect waste diluent / reagent from the priming process.

[0033] In another embodiment contemplated in this disclosure, a small amount of measured whole blood may be mixed manually with an imprecise amount of diluent / reagent in a sample preparation device. For example, a known amount of sample and an imprecise diluent / reagent may be placed in a sample tube and gently rocked back and forth several times. The entire mixed volume is then transferred to a test cartridge having an imaging chamber large enough to accommodate the entire mixed volume by using a transfer pipette or similar device. According to an embodiment of the present invention, if all blood cells in the imaging chamber are counted (directly or by statistical sampling), the blood cell concentration per unit volume can be determined without needing to know the volume of diluent / reagent, the dilution ratio, or the volume of the mixed sample in the imaging chamber, or the volume of the imaging chamber that the mixture occupies. One disadvantage of this embodiment is that it is practically difficult to measure a small amount of sample, for example 1 μL. If a larger amount of sample is chosen, such as 10 μL, it will take longer to count all blood cells and a relatively large imaging chamber will be required. For a sample size of 10 μL and a dilution ratio of 50:1, a 500 μL imaging chamber would be required. An alternative embodiment would be to take a 10 μL sample, mix it with a precise amount of diluent, and then take a portion of the mixture and transfer it to an imaging chamber large enough to accommodate that portion of the mixture. If all the blood cells in the mixture were counted, the number of blood cells per unit volume could be easily determined since the dilution ratio is known.

[0034] The diluents / reagents, either preloaded in the test cartridge or provided in a separate sample preparation device or by a supply module, are in a ready-to-use format. For CBC analysis, the reagents may include a membrane-permeable dye, such as Acridine Orange, which differentially stains DNA and RNA of blood cells in whole blood. Other stains known to those skilled in the art, such as cyanine dyes, may also be used to stain blood cells. In an alternative configuration, the stain may be provided in the form of a dry reagent, with a diluent that is mixed with the dry reagent as needed. Multiple stains may be included in a combination reagent. In one embodiment, the reagent may include an antibody conjugated to a detectable label that targets a particular blood cell or a particular antigen associated with the blood cell. The detectable label may be a dye. The detectable label may be a fluorescent dye, a quantum dot, a colloidal metal such as gold, silver, or platinum, or other detectable construct known in the art. Detectable labels may also be used to detect antibodies specific for blood cells, such as CD3, CD4, CD14, CD16, CD19, CD34, CD45, CD56, or any other enumerated cluster of differentiation markers. Detectable labels may also be used to detect bacterial or parasitic pathogens, platelets, circulating tumor cells, white blood cells, stem cells, or any combination thereof.

[0035] In other embodiments, the liquid reagent may contain a surfactant such as polysorbate or sodium dodecyl sulfate (SDS), an anticoagulant such as EDTA, and / or a sphering agent such as a zwitterionic surfactant to isovolumically reshape red blood cells to facilitate blood cell size measurements and calculation of mean corpuscular volume (MCV).

[0036] The system according to the invention can exhibit better quantitative accuracy than manual microscopic analysis using a manual hemocytometer or similar device, which tends to be limited by sample preparation variability and limited counting statistics. In an embodiment of the invention, sample preparation is improved by eliminating significant operator fluid handling steps and by automating all dilution steps. Any errors in sample dilution are irrelevant because all blood cells and platelets are counted in the entire metered sample volume.

[0037] The system according to the invention can also save time that would otherwise be allocated to manual hemacytometer slide preparation, set-up time, and microscope focusing, which may limit the number of blood samples that can be analyzed. Through automation, the image acquisition and analysis speed can be significantly improved, allowing more blood cells to be analyzed and counted. This can improve the counting statistics and overall accuracy of the system.

[0038] The system according to the invention can also expand the capabilities of blood cell counting methods by allowing point-of-care testing of CBC, i.e., testing near the patient, allowing for immediate clinical decision making. The system can be operated by relatively low skilled personnel. The analyzer can be designed to be inexpensive to manufacture and easily serviced, allowing for easier deployment at clinical sites, such as at the patient's bedside, in clinics, and in emergency departments.

[0039] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0040] In the drawings, like reference characters generally refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. In the following description, various embodiments of the invention are described with reference to the following drawings, in which: [Brief description of the drawings]

[0041] [Figure 1] 1 is a perspective view of an exemplary test cartridge positioned to collect a drop of blood from a patient's finger. [Diagram 2] Figure 2A is a perspective view of an exemplary test cartridge with the cover shown in an open position ready to receive a biological sample, and Figure 2B is a perspective view of the test cartridge shown in Figure 2A with the cover shown in a closed position ready for analysis. [Diagram 3] FIG. 2 is a cutaway view of a blood cell analyzer showing the internal components with a test cartridge inserted. [Figure 4] FIG. 1 is a plan view of an exemplary test cartridge of the type that contains reagents for performing a test. [Diagram 5] FIG. 2 is a plan view of an exemplary test cartridge of the reagent-free type. [Figure 6] 1 is a perspective view of an exemplary reagent supply module showing a test cartridge ready for mating with the module. [Figure 7] 7A and 7B are bottom and side views of a rotary valve stem with a metering chamber formed in a face of the valve stem, with a through conduit serving as the metering chamber. [Figure 8A] 1 is a top view of an exemplary test cartridge showing a sample of whole blood placed in the input port area. [Figure 8B] 8B is a top view of the test cartridge of FIG. 8A showing initial transfer of sample and reagents with the rotary valve in a first open position. [Figure 8C] 8C is a top view of the test cartridge of FIG. 8B, with the valve in a second, open position. [Figure 8D]FIG. 8D is a top view of the test cartridge of FIG. 8C showing the sample and reagents within the imaging chamber. [Figure 8E] FIG. 8E is a plan view of the test cartridge of FIG. 8D, showing the sample and most of the reagents positioned within the mixing chamber. [Figure 8F] FIG. 8F is a top view of the test cartridge of FIG. 8E showing all samples and reagents positioned within the imaging chamber and the valve in the final closed position. [Figure 9] FIG. 9B is a side view showing a cross section of the passive mixing chamber taken along line 9-9' of FIG. 8E. [Figure 10] FIG. 13 is a plan view showing an alternative test cartridge including a sample input port and an imaging chamber. [Figure 11] 4 is a flowchart showing the operation of the blood cell analyzer. [Figure 12] FIG. 1 is a top view of a test cartridge including a sample cup and a serpentine imaging chamber. [Figure 13] FIG. 1 is a top view of a test cartridge including a serpentine imaging chamber. [Figure 14] FIG. 16 is a plan view showing a hematology analyzer that uses the single-test disposable test cartridge shown in FIG. 15. [Figure 15] FIG. 2 is a top view of the test cartridge showing the mixing bowl, sample cup, and serpentine imaging chamber. [Figure 16A] 1A-1C are bright field and fluorescent images showing the same blood cells collected according to the present invention. [Figure 16B] 1A-1C are bright field and fluorescent images showing the same blood cells collected according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] FIG. 1 shows a test cartridge 100 positioned to collect a drop of blood 120 from a patient's finger. The test cartridge is held under the drop 120 such that the dripping drop 120 contacts an input port 130 of the test cartridge 100. The input port 130 includes a recessed area or opening that may be coated with an anticoagulant and may have a surface treatment or features, such as small columns, that enhance surface retention for collecting and holding the blood sample. In an alternative embodiment, the blood sample 120 may be collected intravenously and introduced to the input port 130 by a transfer pipette or capillary tube. The transfer pipette or capillary tube may include an anticoagulant coating according to a desired workflow. The amount of blood or other biological sample placed in the input port 130 is sufficient to visually fill the recessed sample area, but is not measured.

[0043] 2A shows the test cartridge 100 with the closure 135 shown in an open position, providing access to the input port 130. The closure 135 is adapted to slide relative to the test cartridge 100, allowing for movement to facilitate placement in different positions. The closure 135 may have a stop or other positioning mechanism. After the biological sample is collected in the input port 130, the closure 135 may be moved to the position shown in FIG. 2B to cover the input port 130. The closure 135 may be moved by a user before being inserted into the analyzer, as shown in FIG. 3. Alternatively, the closure 135 may be moved by manipulation within the blood cell analyzer. Alternative embodiments of the closure 135 may include graphics, identification information, or instructions for use to the user. Although the closure 135 is shown as a sliding component, other means of closing the input port 130 are envisioned, including a hinged upwardly moving cup, a small surface cover that pivots away from the input port 130 and back to cover the input port 130, or an adhesive component that adheres to the input port 130 or the area surrounding it. In all cases, the closure 135 includes a vent or airway to the input port that allows the blood sample to travel into the test cartridge 100.

[0044] FIG. 3 is a cutaway view of an exemplary blood cell analyzer 200 with a test cartridge 100 positioned for introduction into the analyzer by an operator. From the outside of the blood cell analyzer 200, the housing 206, the user interface screen 208, the printer 212, and the cartridge loading door 217 are visible. When the cartridge loading door 217 is open, the test cartridge 100 can be placed on a cradle 220 of an xy stage 225 configured to receive the test cartridge 100 from a user. The cradle 220 mechanically aligns the cartridge to facilitate connection between the analyzer and the cartridge. For example, a mechanical presser foot 230 may be placed in contact with a flexible surface of the test cartridge to provide mechanical pressure to the packaged and loaded reagents. Some embodiments of the blood cell analyzer 200 may utilize a reagent supply module 470, as further described with reference to FIG. 6. The reagent supply module 470 may be mounted on the xy stage 225 and has a receiving area 473 (see FIG. 6) that aligns the test cartridge 402 with the reagent module 470 .

[0045] The valve driver 235 can be positioned to operate the rotary valve on the test cartridge. The vacuum / pressure pump 240 provides negative or positive pressure to a manifold 245 that interfaces with the test cartridge 100 when the test cartridge 100 is placed in the hematology analyzer, as described below. The hematology analyzer 200 further includes a system controller 250 that controls the movement of fluid within the test cartridge by activating the vacuum / pressure pump 240, by moving the mechanical presser foot 230, or by operating the valve driver 235 according to a preprogrammed sequence. A monitoring camera 255, positioned to capture digital images of the fluid within the cartridge, provides feedback to the system controller 250. The monitoring light source 256 can be a ring illuminator that surrounds the lens of the monitoring camera 255. Information from the monitoring camera 255 is used to control the movement of fluids, to position the rotary valve, and to provide feedback to verify critical steps.

[0046] Also shown in Figure 3 are components of the blood cell analyzer 200, including the automated microscope. At the base of the analyzer, a bright field light source 260 provides illumination through the test cartridge to an objective lens 265 operably coupled to a focusing mechanism. At the top of the analyzer, a fluorescent light source 270 provides illumination through a dichroic mirror 277 to fluorescently excite the sample. At the rear of the analyzer, a digital camera 280 captures images of the test cartridge 100 and transmits them to an image processor / computer 290. In some embodiments, the blood cell analyzer may further include a photometric light source 293 and a light intensity detector 295 that measure light transmission at one or more wavelengths within the chamber of the test cartridge 100, such as for measuring hemoglobin, as described further below.

[0047] FIG. 4 illustrates an exemplary test cartridge 401 of the type that includes liquid reagents stored in a blister pack 417 for performing a test. The test cartridge 401 has an input port 407 for receiving a sample, a passive mixing chamber 405 for mixing the sample with a diluent / reagent, and an imaging chamber 403 for acquiring an image of blood cells in the sample and diluent / reagent mixture for analysis. In this embodiment, the photometry chamber 409 may be filled with whole blood to perform an absorbance measurement to determine the concentration of a particular analyte in the sample, such as hemoglobin. A rotary valve 415 provides fluidic connections between various fluidic channels, vents, and ports, such as sample driver port 411, vent 423, and mixture driver port 429, as described in FIGS. 8A-8F.

[0048] 5 illustrates an exemplary test cartridge 402 of the type that does not include diluents / reagents. Many of the functional components are the same as those illustrated with reference to test cartridge 401, but instead of including diluents / reagents, test cartridge 402 has a reagent input port 460 adapted to be connected to an external diluent / reagent source. Test cartridge 402 may be used in embodiments where the diluents / reagents required for the analysis may be too costly to package separately or may require refrigerated storage. In such embodiments, the diluents / reagents may be provided from a source within hematology analyzer 200 or from a reagent supply module.

[0049] 6 illustrates an exemplary reagent supply module 470 positioned to receive a test cartridge 402. The reagent supply module 470 includes a receiving area 473 for docking the test cartridge 402 and contains a container for holding diluent / reagent, a reagent metering pump adapted to pump the diluent / reagent, and a reagent output port 475. The reagent output port 475 is constructed of a suitable shape and / or elastomeric material that ensures a fluid-tight connection to the reagent input port 460 of the test cartridge 402 when the test cartridge is docked to the reagent supply module 470. The reagent supply module 470 has an opening 477 that is appropriately sized to allow the monitoring camera 255 (FIG. 3) to image the rotary valve 415. Additionally, a window 478 of the reagent supply module 470 is constructed to align with the photometering chamber 409 of the test cartridge. Window 478 allows photometric light source 293 and light intensity detector 295 (FIG. 3) to make absorbance measurements on the fluid in photometric chamber 409 .

[0050] In one embodiment, the size of the containers in the reagent supply module 470 is sufficient to provide diluent / reagent for diluting and / or staining from 10 to about 100 samples with a diluent / reagent to sample ratio of 10:1 to about 250:1. The reagent supply module 470 may further include a self-priming mechanism for priming liquid reagents and eliminating air bubbles. In such an embodiment, the reagent supply module 470 may include a chamber for collecting waste reagent from the priming process. When the test cartridge 402 is docked with the reagent supply module 470, the combined components perform the same function as the test cartridge 401, except that the reagent supply module 470 replaces the blister pack 417. Inside the hematology analyzer 200, the vacuum / pressure pump 240 connects to the sample driver port 411 and the mixture driver port 429 through the manifold 245. The interface connections between manifold 245 and these ports are constructed of appropriate geometry and / or elastomeric materials to ensure an airtight connection so that system controller 250 can control the movement of fluids within the test cartridge (see FIG. 3). In such an embodiment, presser foot 230 is not required.

[0051] The only volume that is accurately measured is the metered volume of the original biological sample. Various means for measuring small amounts of liquid are well known in the art. Two devices well suited for use in this application are shown in Figures 7A and 7B. Figure 7A shows the face of a cylindrical valve stem 485 of a rotary face valve. A metering chamber 483 is formed in the face by a very precise manufacturing process such as injection molding. The chamber 483 is narrow and tubular in shape and is centered in the face of the cylindrical stem 485. A slot 487 at the top of the stem 485 acts as a valve indexer indicating the position of the valve stem 485. An auxiliary connector 421 having a circular shape is also formed in the face of the valve stem 485. When incorporated into a rotary valve 415 (Figures 4 and 5), the metering chamber 483 can connect between ports of the valve spaced 180 degrees apart, and the auxiliary connector 421 connects between other ports spaced 60 degrees apart. 8A-8F, system controller 250 can control fluid movement by rotating valve stem 485 and by positioning the valve via valve indexer 487 according to a preprogrammed sequence. Thus, in a first position, metering chamber 483 can be connected to input port 407 (FIGS. 4 and 5) and filled with a biological sample, and then by rotating valve stem 485, the volume contained within metering chamber 483 can be isolated and transferred for analysis.

[0052] Figure 7B is a side view of a valve stem 485' in which the metering chambers are formed as through conduits 413 in the tapered seat of the valve stem 485'. The through conduits 413 can connect with fluid channels of the rotary valve 415 spaced 180 degrees apart. Also shown in Figure 7B is an auxiliary fluid connector 421' that connects to adjacent fluid channels spaced 60 degrees apart.

[0053] When incorporated into a rotary valve 415 (FIGS. 4 and 5) having a tapered seat that receives a valve stem 485', the through conduit 413 can be connected to an input port 407 (FIGS. 4 and 5), filled with a biological sample, and then by rotating the valve stem 485', the volume of sample contained within the through conduit 413 can be isolated and transferred for analysis. FIG. 7B also shows an auxiliary fluid connector 421' that fluidly connects to an adjacent fluid channel of a test cartridge according to the position of the valve's indexer 487'. It will be appreciated that the rotary face valve of FIG. 7A and the tapered seat valve of FIG. 7B are alternative embodiments for isolating samples and controlling fluid paths. Thus, in the following description, references to the metering chamber 483 of the rotary face valve will be equally applicable to the through conduit 413 of the tapered seat valve.

[0054] 8A-8F and with reference to FIG. 3, a sequence of operations is shown that allows the blood cell analyzer 200 to perform automated microscopic blood cell analysis on a biological sample without the interaction of a skilled operator. In FIG. 8A, a sample is shown deposited in the input port 407, which is in fluid communication with the rotary valve 415. As shown in FIG. 8A, the axis 485 (FIG. 7A) of the rotary valve 415 is in a first position in which the metering chamber 483 (FIG. 7A) is aligned with the sample input port 407 and the sample driver port 411. A vacuum supplied by the analyzer to the sample driver port 411 draws the sample from the input port 407 into the metering chamber 483 and into the photometry chamber 409. Once the photometry chamber 409 is filled with sample, the system controller 250 (FIG. 3) collects absorption data from the undiluted sample using the photometry light source 293 (FIG. 3) and the light intensity detector 295 (FIG. 3). As will be appreciated by those skilled in the art, appropriate selection of light wavelengths, and chamber geometry, and analysis of the light passing through the biological sample can be used to determine the concentration of a particular analyte in the sample, such as hemoglobin.

[0055] By way of example, and with reference to FIG. 8B, cartridge 401 is shown with diluents / reagents contained in blister pack 417. When rotary valve 415 is positioned such that metering chamber 483 is aligned with input port 407 and metering chamber 409, auxiliary connector 421 provides a fluid communication path between blister pack 417 and vent 423. Pressure is applied to blister pack 417 by presser foot 230 (FIG. 3). When the diluent / reagent is released and flushed through auxiliary connector 421, priming the channel and removing air bubbles through vent 423.

[0056] 8C shows rotary valve 415 rotated 60 degrees counterclockwise to a second position that isolates a predetermined amount of sample in metering chamber 483. In this second position, stem 485 of rotary valve 415 is positioned such that metering chamber 483 is in fluid communication with blister pack 417 and serpentine imaging chamber 403.

[0057] In Figure 8D, the rotary valve 415 is shown in the same position as in Figure 8C, but following the action of the presser foot 230 applying pressure to the blister pack 417. As indicated by the shaded area, diluent / reagent from the blister pack 417 and the isolated sample 493 from the metering chamber 483 are transferred into the imaging chamber 403. A minimum amount of reagent that is three times the volume of the through conduit 413 is required to flush the entire sample out of the rotary valve 415. Depending on the analysis to be performed, a sufficient amount of reagent is forced through the rotary valve 415 to completely flush out the isolated sample and achieve the desired approximate dilution ratio.

[0058] In FIG. 8E, the rotary valve 415 is shown rotated 120 degrees counterclockwise from the previous position shown in FIG. 8D to a third position, with the auxiliary connector 421 aligned with the mixture driver port 429 and the imaging chamber 403. The vacuum / pressure pump 240 (FIG. 3) of the blood cell analyzer 200 provides pressure to the mixture driver port 429, forcing all of the sample and diluent / reagent mixture out of the imaging chamber 403 and into the passive mixing chamber 405. As the mixture enters the passive mixing chamber 405, air within the chamber is vented through the vent port 433. Once all of the sample and diluent / reagent mixture has been transferred to the passive mixing chamber 405, the vacuum / pressure pump 240 applies a controlled vacuum to the mixture driver port 429, causing the mixture to be drawn back into the imaging chamber 403. The preprogrammed sequence of pushing the mixture into the passive mixing chamber 405 and pulling it back into the imaging chamber 403 is repeated to achieve a final mixture 495 that is free of clumping and overlapping of blood cells after they settle to the bottom of the imaging chamber 403. In the final movement of the mixture 495, it is positioned entirely within the imaging chamber 403 as shown in FIG. 8F. In most cases, the inventors have found that pushing and pulling the sample and diluent / reagent into and out of the mixing chamber 405 is sufficient to obtain a homogenous mixture. Furthermore, the mixture remains substantially homogenous when transferred into the serpentine imaging chamber 403. It should also be noted that the mixing chamber 405 can be located at the start of the imaging chamber 403.

[0059] 8F shows the final step of the sample preparation sequence. At this point in the preprogrammed sequence, the entire final mixture 495 has been extracted from the passive mixing chamber 405 and positioned within the imaging chamber 403. Once this position is achieved, the rotary valve 415 is rotated counterclockwise approximately 30 degrees to the position shown in FIG. 8F such that it is no longer in fluid communication with any of its fluid channels, thereby preventing further fluid communication with the imaging chamber 403 and preventing further movement of the final mixture 495.

[0060] FIG. 9 shows a cross-section of a passive mixing chamber 405. The chamber is called "passive" because, as shown, it does not contain any active mixing elements such as beads or spin bars. While such devices may be used in some embodiments, the inventors have found that an appropriately sized chamber such as that shown in FIG. 9 is simpler and provides superior mixing of the sample and reagents. In operation, diluent / reagent and sample 493 are driven by vacuum / pressure pump 240 (FIG. 3) to move in and out of the chamber through mixing chamber opening 497. As liquid enters the chamber, air in the chamber is drawn out through the vent port 498. The mixture escapes through vent port 433. A cross section of passive mixing chamber 405 shows a wall geometry that smoothly increases in size from bottom to top so that the mixture entering from below expands into a larger volume. Chamber 405 may have asymmetrical sloping walls 484 and 491 to promote mixing of the sample and reagents and to remove bubbles from the mixture. After all of the mixture has entered the chamber, air bubbles may be introduced into the chamber through mixing chamber opening 497 by vacuum / pressure pump 240. These air bubbles further promote mixing and then escape through vent port 433. The selection of materials used to fabricate passive mixing chamber 405 should take into account the wettability of the particular diluent / reagent utilized in test cartridge 401. The nature of the material should ensure, among other requirements, that when vacuum / pressure pump 240 empties the chamber through mixing chamber opening 497, the surface tension of the liquid will pull back any liquid in contact with the side walls of the chamber.

[0061] FIG. 10 shows a test cartridge 900 with an imaging chamber 903 having a sample input port 950 at one end and a vent 953 at the opposite end. A user of the test cartridge 900 collects a known small amount of whole blood and manually mixes it with diluent / reagents in a separate single-use sample preparation device (not shown). Once mixed, the entire mixed volume is injected into the sample input port 950 at a controlled rate, with the blood cells filling the imaging chamber evenly. Air escapes through the vent 953, allowing the sample and diluent / reagent mixture to fill the imaging chamber 903. The configuration of the imaging chamber is essentially the same as the imaging chamber as described above and shown in FIGS. 8A-8F, except that the volume of the imaging chamber must be sufficient to contain all of the sample and diluent / reagent mixture. The test cartridge 900 can be placed into the analyzer 200 (FIG. 3) to count all blood cells in the sample and diluent / reagent mixture and begin analysis at step 560 of FIG. 11, as described below.

[0062] With attention now to FIG. 11, the general operation of the blood cell analyzer 200 configured to provide a "CBC with differential" analysis will now be described with reference to the test cartridge 401 shown in FIGS. 8A-8F and the blood cell analyzer 200 shown in FIG. 3. To obtain a blood sample from a patient presented in box 500, a user first obtains a new test cartridge 401 in box 505 and opens it to expose it to the input port 407. Blood from a finger prick is applied as shown in FIG. 1 in box 510 and the input port 407 is covered. The user inserts the test cartridge into the blood cell analyzer 200 in box 515. The test cartridge is moved into the analyzer where mechanical and fluidic connections are made between the analyzer and the cartridge as described above with reference to FIG. 3. In the first step of the analysis, the sample is drawn into the metering chamber through the photometric chamber 409 and into the photometric chamber 409 (FIG. 8A). In box 520, the absorbance of the blood is measured. Data from the absorbance measurement is used to determine the hemoglobin concentration. The sample in the metering chamber 483 is imaged using the monitoring camera 255 in box 530 and analyzed in box 535 to ensure the metering chamber was properly filled. If an error is detected, the analysis is terminated in box 537 and the user is alerted of the error and instructed to remove the cartridge and reject the test.

[0063] If the through conduit 413 is properly filled, then the diluent / reagent channels are primed, as described above with reference to Figure 8B, at box 540. The rotary valve 415 is then turned to the position shown in Figure 8C to isolate the sample, allowing the diluent / reagent to flush the metered blood volume out of the through conduit 413, at box 545, while being imaged by the monitoring camera 255. The transfer continues until the monitoring camera 255 confirms that the diluent / reagent and sample have nearly filled the imaging chamber, as shown in Figure 8D.

[0064] Once a sufficient amount of diluent / reagent has been transferred, the rotary valve 415 is positioned as shown in Figure 8E and the entire volume of sample and diluent / reagent are mixed at box 550. The entire volume 495 is transferred to the imaging chamber at box 555 and the rotary valve 415 is positioned as shown in Figure 8F. Note that by transferring the entire volume 495 of the mixed sample, the metered volume of blood from the original sample plus the unmetered volume of diluent / reagent is all positioned in the imaging chamber at box 555.

[0065] If a test cartridge 400 was used, it is inserted into the blood cell analyzer 200 in step 560 and analysis begins. Analysis of the test cartridge 401 or 402 continues in step 560 as the xy stage 225 moves the test cartridge 401 to obtain bright field and fluorescent images of the entire imaging chamber 403 in box 560. In an alternative embodiment, the objective lens 265 and / or the digital camera 280 are moved and the test cartridge 401 remains stationary. In yet another embodiment, the objective lens 265 has a field of view sufficient to acquire the entire imaging chamber 403 without movement. The two digital images of each physical frame of the imaging chamber are transferred to the image processor / computer 290 in box 565. One image obtained using bright field optics can be compared to the other image obtained using fluorescent optics to identify red blood cells, white blood cells, and platelets. By further analyzing the size and internal structure of the white blood cells, pattern recognition can be used to identify white blood cell subtypes.

[0066] At box 570, mature red blood cells can be differentiated from reticulocytes and nucleated red blood cells by comparison of bright field and fluorescent images. The concentration (cells per unit volume) can be determined by dividing each cell count by the known volume of the metering chamber 483. The planar size of the red blood cells can be converted to mean corpuscular volume (MCV) by using a sphering agent. Combining the red blood cell count with the MCV and the volume of the metering chamber 483 allows for the calculation of the hematocrit (HCT) and red blood cell distribution width (RDW). The separately measured HGB from box 525 can be used in combination with the RBC count for further calculations to obtain the mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin content (MCHC).

[0067] At box 575, the measurement results are compared to previously defined limits and ranges for the particular patient population to determine whether the results are within or outside of normal expected ranges. According to this determination, at box 580, results within the normal range are reported, and at box 585, results outside the normal range are reported.

[0068] As mentioned above, another embodiment of the present invention is to perform a CBC on a known or measured volume of a diluted sample. In this embodiment, all blood cells and platelets in the known volume of the diluted sample are counted. If the volume of the diluted sample and the dilution ratio are known, the number of blood cells and platelets per unit volume of the sample can be determined. A hematology analyzer can be provided to perform a CBC on a known volume of the diluted sample using a single-use disposable test cartridge. The length and depth of the imaging chamber depends on the dilution ratio and volume of the known diluted sample. For example, a 20 μL whole blood sample may be diluted 50:1 to produce a 1000 μL diluted sample. 20 μL of the diluted sample may then be removed and analyzed. All blood cells and platelets in the diluted sample are counted directly or by statistical expression. A known diluted volume of 20 μL is equivalent to .4 μL of whole blood. To accommodate the entire known volume of the diluted sample, the volume of the imaging chamber must be at least 20 μL.

[0069] The dilution ratio must be sufficient to prevent clustering or overlapping of blood cells as they settle to the bottom of the imaging chamber. The dilution ratio also depends on the depth of the imaging chamber as discussed above. The volume of diluted blood must be sufficient to contain enough white blood cells to be meaningfully representative of the whole blood sample. For example, the average white blood cell count in whole blood of a healthy patient may be about 5000 per microliter. In .4 μL of whole blood, there will be about 2000 white blood cells. However, in a sick patient, or a patient being treated with chemotherapy, the white blood cell count may be as low as 500 per microliter. In this case, the white blood cell count in the .4 μL diluted sample would be about 200, an inadequate white blood cell count to be clinically significant. In this case, a larger volume of diluted sample may be desirable. However, as the volume of the diluted sample increases, the time to image and count all blood cells in a known volume of the diluted sample increases.

[0070] A hematology analyzer utilized to perform a CBC on a sample of known dilution volume includes an automated microscope that images blood cells in an imaging chamber of a test cartridge, similar to that described above and shown in FIG. 3, except that it need not have a presser foot and valve driver. In one embodiment, the dilution step may be performed manually with a pipette and mixing tube or beaker and outside the analyzer, in which case the dilution ratio will be a known ratio. A known volume of the mixture may be placed in a sample cup 701 of FIG. 12 on a test cartridge 703 having a serpentine imaging chamber 709. The sample cup may be fluidly connected to the imaging chamber 709 by a channel 704 at one end of the imaging chamber 709. At the opposite end of the serpentine chamber 709 is a vent hole 707. When the test cartridge is inserted into the analyzer, the vent hole 707 interfaces with the analyzer's vacuum / pressure source. When a vacuum is applied to the vent hole 707, the diluted sample is drawn into and positioned within the imaging chamber 709 such that its entire volume is located within the imaging chamber. The diluted sample is also forced back into the sample cup by pressure applied to the vent hole and then back into and positioned within the imaging chamber for purposes of mixing the diluted sample in a manner similar to that described above with reference to the mixing chamber 405 of FIG. 8E. The sample cup may be of the same shape and format as the mixing chamber 405 of FIG. 8E to facilitate mixing if necessary. For example, if a known volume of diluted sample is placed in the sample cup and a test cartridge is not inserted into the analyzer to perform an immediate blood cell analysis, blood cells in the diluted sample may accumulate at the bottom of the sample cup. In this case, mixing may be required. Once the diluted sample is positioned within the imaging chamber, the analyzer may perform a CBC on the diluted sample as described above.

[0071] Alternatively, a measured volume of sample and a measured volume of diluent / reagent may be mixed manually, and a portion of the mixture with a known volume may be inserted into a test cartridge having a serpentine imaging chamber as shown in Figure 13 at a controlled flow rate that prevents cell crowding and ensures uniform distribution. The dimensions of the serpentine path are chosen according to the dilution ratio, the known volume of the mixture, and the guidelines discussed above. Each cell in the known volume of the mixture may be counted and analyzed as discussed above.

[0072] In another embodiment, the dilution and sample preparation steps may be performed by the analyzer utilizing a probe to aspirate a whole blood sample, a shear or face valve to isolate a predetermined amount of sample, a source of diluent / stain, a syringe pump to meter and dispense a known amount of diluent / stain in a mixing bowl that mixes the sample and diluent / stain, solenoid rocker or pinch valves to control fluid movement, a vacuum and pressure source, and a disposable, single-use test cartridge. The shear valve is in fluid communication with the analyzer probe that aspirates the blood sample. The sample may be drawn through the probe into the shear valve, which turns the shear valve to capture a predetermined amount of sample. The shear valve may also be coupled to a pressure source and The probe is rotated into a position in fluid communication with the mixing bowl. The isolated blood sample is forced into the mixing bowl by a pressure source. A syringe pump is in fluid communication with the diluent / stain source and the mixing bowl. The syringe pump dispenses a predetermined amount of diluent / stain into the mixing bowl. The blood sample and diluent / stain can be mixed in the bowl by forcing air through the probe, bubbling air through the mixture. Once mixed, a portion of the mixture may be drawn into a metering chamber of known volume and in fluid communication with the mixing bowl. An optical edge detection sensor is used to control the flow of the mixture into the metering chamber. The test cartridge shown in FIG. 13 is used as a single-use disposable cartridge with a hematology analyzer. By being inserted into the analyzer, the channel 950 of FIG. 13 at one end of the serpentine imaging chamber 951 interfaces and is in fluid communication with the metering chamber. A vacuum is applied to the opposite end of the serpentine path through vent hole 953 in FIG. 13, and the portion of the diluted sample located in the metering chamber is drawn into and positioned in the imaging chamber at a controlled rate that prevents crowding of blood cells and ensures uniform distribution for CBC analysis as described above. The dimensions of the serpentine path depend on the dilution ratio, the volume of the mixture portion, and the guidelines above. One disadvantage of this configuration is that the shear valve, metering chamber, mixing bowl, metering chamber, and connecting fluid channels must be flushed after each sample. Such analyzers also require frequent calibration and maintenance.

[0073] In another embodiment, the dilution and metering steps may be performed with a test cartridge having a mixing bowl and with a diluent sample cup on the test cartridge. The analyzer includes a sampling probe, a diluent / stain reservoir, a precision diluter syringe pump, and a wash station. In this case, the probe may be attached to a transfer arm mounted on the base of the hematology analyzer 200 of FIG. 3 that can move vertically in the z-direction relative to the stage 225 of FIG. 3. The transfer arm can move horizontally along a linear axis so that it can vertically align with the wash station, or the diluent / reagent reservoir, or the sample container. A schematic of the analyzer is shown in FIG. 14. The dilution step is as follows: The probe 801 of FIG. 14 moves along the linear axis 803 until it is vertically aligned with the diluent / stain reservoir 805. It then moves vertically downward until the tip of the probe is immersed in the diluent / stain 805. It then aspirates a known amount of diluent / stain, for example 1000 μL. The probe moves upward and horizontally along axis 803 until it is aligned with the sample container 807. After aspirating 3 μL of air, it moves vertically downward until the tip of the probe is fully immersed in the sample in the sample container 807. The analyzer then aspirates 20 μL of sample, such as whole blood, from the sample container, and then moves the probe upward above the sample container where it aspirates another 3 μL of air bolus. The probe then moves along linear axis 803 until it is vertically aligned with the mixing bowl 721 of FIG. 15 on the test cartridge 723. The probe is then lowered vertically downward until the tip of the probe is just above the bottom of the mixing bowl 721. The analyzer then dispenses the blood sample and diluent / stain into the mixing bowl 721 on the test cartridge 723. This sequence of steps ensures that the entire aspirated blood sample is washed out of the probe by the diluent / stain reagent and into the mixing bowl.The analyzer may mechanically mix the blood and diluent, such as by moving back and forth along axis 803, or by bubbling air through the mixture, or by aspirating the mixture and redistributing it into a mixing bowl, or by other methods. After the sample and diluent / stain are mixed, the analyzer aspirates 20 μL of the mixture, which corresponds to .4 μL of undiluted sample. The probe is then moved upwards and along linear axis 803, FIG. 14, until the probe is aligned with diluted sample reservoir 728, FIG. 15. The probe is lowered down and 20 μL of the sample and diluent / stain mixture is dispensed into sample cup 728, FIG. 15, which is in fluid communication with one end of serpentine pathway 719 through channel 731. The mixture may be forced through the serpentine path of the imaging chamber 719 at a controlled rate by a vacuum source of the analyzer, which interfaces with a vent hole 717 at the end of the imaging chamber opposite the sample cup 728. Alternatively, after the sample is dispensed, a probe may be positioned in the vent hole 717, which may include a sealing O-ring, and the analyzer draws air through the probe at a controlled rate, such as 2 μL per second, to prevent cell crowding and ensure uniform distribution. The analyzer positions the entire sample and diluent / stain mixture in the imaging chamber 719. The dimensions of the serpentine path depend on the dilution ratio, the volume of the mixture, and the considerations above. Another variation utilizes a test cartridge that does not include a sample cup, in which case the probe may draw 20 μL of diluted sample and then interface directly with the fluidic channel 731 to dispense the mixture directly into the serpentine path of the imaging chamber 719 at a controlled rate.

[0074] After aspirating or dispensing blood or diluent / stain, or a mixture of sample and diluent / stain, the probe may be washed in wash station 809 of Figure 14 to remove any solution adhering to the sides of the probe. Although this process has not been described above, one of ordinary skill in the art related to chemical analyzers or XY dispensing fluid mechanisms will understand the practices and procedures for doing so.

[0075] One advantage of this embodiment is that shear valves and fluid tubing and their cleaning are eliminated, as well as the interconnection of sample-to-sample fluid channels. The need for pinch valves and / or solenoid valves is also reduced or may be eliminated.

[0076] The analyzer may process samples in parallel by performing a CBC imaging analysis on a known diluted sample in the imaging chamber of one test cartridge while at the same time the analyzer dilutes another sample and places it in the imaging chamber of a second test cartridge. This can be done to increase the throughput of the analyzer. EXAMPLES

[0077] EXAMPLES

[0078] Information from brightfield and fluorescence optics Figure 16 shows images collected using a test device according to the present invention. The fluorescent stain Acridine Orange (AO) was used to differentially stain DNA and RNA of blood cells in a whole blood sample. The visual images in Figure 16 were obtained using an Olympus 20X×0.4NA objective lens 265 and a Basler 5 MP digital camera 280.

[0079] Excitation for the second row brightfield images was provided by a white light brightfield light source 260. Excitation for the third row fluorescent images was a 455 nm blue fluorescent light source 270. White blood cells have significant RNA and DNA and are therefore visible in the fluorescent images with green and orange structures. The size and shape of the green nuclear structures and the overall size of the white blood cells can be used to differentiate them into subpopulations identified by the names in the first row. In particular, the basophil and eosinophil subpopulations of white blood cells have characteristic properties in the bright field images due to the presence of large granules in the cytoplasm. Thus, embodiments of the present invention utilize analysis of both bright field and fluorescent images to differentiate between subpopulations of white blood cells.

[0080] Platelets also absorb the AO stain, but their size is significantly smaller than either white blood cell and therefore they can be differentiated. Red blood cells lose their nuclei as they mature and therefore have no nuclear material to absorb the AO stain. As a result, they appear in brightfield but not in fluorescent light. As objects, red blood cells can be identified. Immature red blood cells, called reticulocytes and nucleated red blood cells (nRBCs), have the attributes of red blood cells but also exhibit low levels of fluorescence. Embodiments of the present invention utilize these combined attributes to identify and subgroup red blood cells. EXAMPLES

[0081] Statistical Sampling of Imaging Chambers Table 1 shows a comparison of CBC parameters obtained according to the present invention and from an automated hematology analyzer.

[0082] [Table 1]

[0083] Sample: Low WBC count - approximately 2000 / μL (normally 3,000-10,000 / μL) ) Magnification: 20x Number of images: Approximately 10,000 brightfield images, approximately 10,000 fluorescent images Variables: Column 1 - Percentage of total blood cells used in calculation Pair count column - number of image pairs (brightfield and fluorescence) RBC column - total number of red blood cells counted WBC column - the total number of white blood cells counted ROI column - Entire Region of Interest. This is the "effective" number of image frames that the actual sample occupies. A frame completely filled with sample / blood cell is a "1". Partial frames (due to serpentine edges or curved ends) are a fraction of 1 frame (e.g. 0.567).

[0084] RBC / f column - average number of red blood cells per frame (RBC column divided by ROI in column 5) RBC / f(%) column - This is the RBC / frame value at a particular sampling rate divided by the RBC / frame at a sampling rate of 100% (top row). This is an estimate of the accuracy of a particular sampling rate compared to a 100% blood cell count.

[0085] WBC / f column - average white blood cell count per frame (WBC column divided by ROI column) Column 9 WBC / f(%) - This is similar to column 7, but estimates the precision of the sampling rate for white blood cells.

[0086] RBC / WBC column - This is the ratio of RBC / WBC for a particular sampling rate. Result: A small percentage of all frames can provide accurate results. Since a small percentage of all frames are counted, accuracy is maintained down to 1% for red blood cells and 5% for white blood cells.

[0087] Discussion: In these experiments, it took about 1 second to acquire an image pair. Since in this experiment almost 10,000 image pairs were needed to acquire 100% of the sample, this means that the image analysis took 10,000 seconds, about 2.8 hours. The experiments show that the uniformity of the distribution of blood cells across the imaging chamber was good enough to obtain accurate results by counting blood cells in only 5% of the frames. Since the entire sample size (region of interest ROI) is measured, the goal of "counting all blood cells" is achieved, but only 5% of the images need to be analyzed to obtain accurate results. This reduces the image analysis time to about 8 minutes. It is expected that this time will be further reduced with the development of camera and computer processing technology.

[0088] The present invention has been described in connection with a number of specific embodiments. However, numerous modifications that are believed to be within the scope of the present invention will become apparent to those skilled in the art. Accordingly, the scope of the present invention is intended to be limited only by the appended claims. In addition, the order of presentation of the claims should not be construed to limit the scope of any particular term in the claims.

Claims

1. 1. A method for counting and analyzing biological particles in whole blood, including blood cells and platelets, utilizing a blood cell analyzer, a diluent and / or stain, and a test cartridge having an imaging chamber, comprising: a) isolating a known amount of a sample; b) mixing said known amount of sample with a sufficient amount of diluent to form a substantially homogenous mixture of sample and diluent; c) transferring the mixture of sample and diluent into the imaging chamber of the test cartridge; d) acquiring one or more digital images of the mixture in the imaging chamber selected to be statistically representative of the number and distribution of the particles in the imaging chamber; e) counting all of the at least one type of biological particle in said image using image processing software; and f) calculating the number of particles per unit volume of said one type of biological particle in said sample.

2. The method of claim 1 , wherein the acquisition of digital images includes bright field and fluorescent images.

3. The method of claim 1 , further comprising the step of allowing the particles in the mixture to settle on a bottom of the imaging chamber after the step of transporting the mixture into the imaging chamber.

4. 4. The method of claim 3, wherein the imaging chamber has a geometry such that the blood cells do not overlap, crowd, or flow out when the particles accumulate on the bottom of the imaging chamber.

5. The method of claim 1 , further comprising the step of analyzing the biological particles in the acquired images using pattern recognition software.

6. The method of claim 5 , further comprising the step of performing a three-way differential of the white blood cells.

7. 6. The method of claim 5, further comprising the step of performing a 5-way differential of the white blood cells.

8. The method of claim 1 further comprising the step of displaying the digital image of the particle.

9. The method of claim 1 , wherein the step of acquiring the digital image includes acquiring an image that includes all particles within the imaging chamber.

10. 10. The method of claim 9, further comprising the step of counting all of the at least one type of biological particles.

11. 2. The method of claim 1, wherein the volume of the sample is between 0.1 μL and 500 μL and the volume of the diluent is between 1 μL and 500 μL.

12. 2. The method of claim 1, wherein the ratio of diluent to sample in said mixture of diluent and sample is from 10:1 to 250:

1.

13. The rate at which the mixture of diluent and sample is transported is such that the mixture remains substantially homogeneous. The method of claim 1 , wherein the speed is a certain speed.

14. 10. The method of claim 1, wherein the mixture of diluent and sample is transported into the imaging chamber at a rate of about 2 μL per second.

15. The method of claim 1 , wherein the imaging chamber has a uniform width and depth, and the imaging chamber has a length to width ratio of greater than 2:

1.

16. 10. The method of claim 1, wherein the imaging chamber has a uniform width and depth, and the imaging chamber has a length to width ratio of about 400:

1.

17. The method of claim 1 , wherein the imaging chamber has a uniform width and is between 0.5 mm and 2.5 mm.

18. The method of claim 1 , wherein the imaging chamber has a uniform depth and width, the width being between 10 and 200 μm.

19. The method of claim 1 , wherein the imaging chamber has a uniform depth and a serpentine shape in plan view.

20. 20. The method of claim 19, wherein an outer turning diameter of the serpentine imaging chamber is approximately twice an inner turning diameter of the serpentine imaging chamber.

21. 2. The method of claim 1, wherein the imaging chamber has a uniform depth and the shape of the imaging chamber in plan view is serpentine in shape, having a width of 1.25 mm, a turn inner diameter of 1.25 mm, a turn outer diameter of 2.5 mm, and a depth of 0.125 mm.

22. The method of claim 1 , wherein the imaging chamber has a helical shape in plan view.

23. The method of claim 1 , wherein the imaging chamber has a contour in plan view that is irregular like a chest wall.

24. The method of claim 1 , wherein the imaging chamber has a geometry such that a distribution of particles in the mixture remains substantially homogenous when the mixture is transferred into the imaging chamber.

25. The method of claim 1 , wherein the step of mixing comprises mixing the sample with a diluent and a stain.

26. 26. The method of claim 25, wherein the step of mixing comprises mixing a stain in dry form.

27. 26. The method of claim 25, wherein the step of mixing includes mixing with a liquid stain.

28. 26. The method of claim 25, wherein the known sample amount is between 0.1 μL and 10 μL and the volume of diluent and stain is between 10 μL and 500 μL.

29. The method of claim 1 , wherein the step of mixing comprises mixing the sample with a diluent, a stain and a sphering agent.

30. 10. The method of claim 1, wherein the steps of separating, mixing, transferring, acquiring, counting, and calculating are applied to a sample comprising at least one of blood cells, platelets, sperm, bacteria, spores, and inorganic particles.

31. 1. A method for counting and analyzing biological particles in whole blood, including blood cells and platelets, utilizing a blood cell analyzer, a diluent and stain, and a test cartridge having an imaging chamber, comprising: a) introducing the sample of whole blood into the test cartridge; b) separating a known amount of sample from the remaining amount of the sample in the test cartridge; c) mixing the known amount of sample in the cartridge with a sufficient amount of diluent to form a substantially homogenous mixture of sample and diluent; d) transferring the mixture into the imaging chamber of the test cartridge; e) acquiring one or more digital images of the mixture in the imaging chamber selected to be statistically representative of the number and distribution of the biological particles in the imaging chamber; and f) deriving a quantitative characteristic of at least one aspect of the distribution of said particles within said imaging chamber based on said image acquired in said acquiring step.

32. 32. The method of claim 31, wherein the step of deriving the quantitative characteristic comprises analyzing the bioparticles in the acquired image using pattern recognition software.

33. 32. The method of claim 31 , wherein the step of deriving the quantitative characteristic comprises counting all of at least one type of biological particle in the image using image processing software and calculating the number of particles per unit volume of the one type of biological particle in the sample.

34. 32. The method of claim 31, further comprising the step of performing a three-way differential of the white blood cells.

35. 32. The method of claim 31, further comprising the step of performing a 5-way differential of the white blood cells.

36. 32. The method of claim 31, wherein the acquiring of digital images includes bright field and fluorescent images.

37. 32. The method of claim 31, further comprising the step of allowing the particles in the mixture to settle on a bottom of the imaging chamber after the step of transporting the mixture into the imaging chamber.

38. 38. The method of claim 37, wherein the imaging chamber has a geometry such that the blood cells do not overlap, cluster, or flow out when the particles accumulate on the bottom of the imaging chamber.

39. 38. The method of claim 37, further comprising the step of analyzing the biological particles in the acquired images using pattern recognition software.

40. 32. The method of claim 31 further comprising displaying the digital image of the particle.

41. 32. The method of claim 31, wherein the step of acquiring the digital image includes acquiring an image that includes all particles in the imaging chamber.

42. 43. The method of claim 42, further comprising the step of counting all of the at least one type of biological particles.

43. 32. The method of claim 31, wherein the volume of sample is between 0.1 μL and 500 μL and the volume of diluent is between 1 μL and 500 μL.

44. 32. The method of claim 31, wherein the ratio of diluent to sample in said mixture of diluent and sample is from 10:1 to 250:

1.

45. 32. The method of claim 31, wherein the rate at which the mixture of diluent and sample is transported is such that the mixture remains substantially homogenous.

46. 32. The method of claim 31, wherein the mixture of diluent and sample is transported into the imaging chamber at a rate of about 2 μL per second.

47. 32. The method of claim 31 , wherein the imaging chamber has a uniform width and depth, and the imaging chamber has a length to width ratio of greater than 2:

1.

48. 32. The method of claim 31 , wherein the imaging chamber has a uniform width and depth, and the imaging chamber has a length to width ratio of about 400:

1.

49. 32. The method of claim 31 , wherein the imaging chamber has a uniform width and is between 0.5 mm and 2.5 mm.

50. 32. The method of claim 31 , wherein the imaging chamber has a uniform depth and width, the width being between 10 and 200 μm.

51. 32. The method of claim 31 , wherein the imaging chamber has a uniform depth and a serpentine shape in plan view.

52. 52. The method of claim 51, wherein an outer turning diameter of the serpentine imaging chamber is about twice an inner turning diameter of the serpentine imaging chamber.

53. 32. The method of claim 31 , wherein the imaging chamber has a uniform depth and the shape of the imaging chamber in plan view is serpentine in shape, having a width of 1.25 mm, an inner turn diameter of 1.25 mm, an outer turn diameter of 2.5 mm, and a depth of 0.125 mm.

54. 32. The method of claim 31 , wherein the imaging chamber has a helical shape in plan view.

55. 32. The method of claim 31 , wherein the imaging chamber has a contour in plan view that is irregular like a chest wall.

56. 32. The method of claim 31, wherein the imaging chamber has a geometry such that a distribution of particles in the mixture remains substantially homogenous when the mixture is transferred into the imaging chamber.

57. 32. The method of claim 31, wherein the step of mixing comprises mixing the sample with a diluent and a stain.

58. 32. The method of claim 31, wherein the step of mixing comprises mixing a stain in dry form.

59. 32. The method of claim 31, wherein the step of mixing includes mixing with a liquid stain.

60. 32. The method of claim 31, wherein the known sample amount is between 0.1 μL and 10 μL and the volume of diluent and stain is between 10 μL and 500 μL.

61. 32. The method of claim 31, wherein the step of mixing comprises mixing the sample with a diluent, a staining agent and a sphering agent.

62. 32. The method of claim 31, wherein the steps of separating, mixing, transferring, acquiring, counting, and calculating are applied to a sample comprising at least one of blood cells, platelets, sperm, bacteria, spores, and inorganic particles.

63. a) introducing the whole blood sample into the test cartridge, and then separating a separate known amount of the sample from the remaining amount of the test cartridge; b) transferring the first known amount of sample to a photometric chamber; c) measuring hemoglobin of the sample in the photometric chamber.

64. 64. The method of claim 63, wherein the step of analyzing the biological particles comprises performing a three-way differential of the white blood cells.

65. 64. The method of claim 63, wherein the step of analyzing biological particles comprises performing a 5-fold differential of the white blood cells.

66. 64. The method of claim 63, wherein the counting step includes counting the red blood cells, the platelets, and the white blood cells.

67. 64. The method of claim 63, wherein the analyzing step comprises measuring mean packed volume.

68. 64. The method of claim 63, wherein the acquisition of digital images includes bright field and fluorescent images.

69. 64. The method of claim 63, further comprising, after the step of transporting the mixture into the imaging chamber, allowing the particles in the mixture to settle on a bottom of the imaging chamber.

70. 70. The method of claim 69, wherein the imaging chamber has a geometry such that the blood cells do not overlap, cluster, or flow out when the particles accumulate on the bottom of the imaging chamber.

71. 64. The method of claim 63, further comprising the step of analyzing the biological particles in the acquired images using pattern recognition software.

72. 64. The method of claim 63, further comprising the step of displaying the digital image of the particle.

73. 64. The method of claim 63, wherein the step of acquiring the digital image includes acquiring an image that includes all particles in the imaging chamber.

74. 74. The method of claim 73, further comprising the step of counting all of the at least one type of biological particles.

75. 64. The method of claim 63, wherein the volume of sample is between 0.1 μL and 500 μL and the volume of diluent is between 1 μL and 500 μL.

76. 64. The method of claim 63, wherein the ratio of diluent to sample in said mixture of diluent and sample is from 10:1 to 250:

1.

77. 64. The method of claim 63, wherein the rate at which the mixture of diluent and sample is transported is such that the mixture remains substantially homogenous.

78. 64. The method of claim 63, wherein the mixture of diluent and sample is transported into the imaging chamber at a rate of about 2 μL per second.

79. 64. The method of claim 63, wherein the imaging chamber has a uniform width and depth, and the imaging chamber has a length to width ratio of greater than 2:

1.

80. 64. The method of claim 63, wherein the imaging chamber has a uniform width and depth, and the imaging chamber has a length to width ratio of about 400:

1.

81. 64. The method of claim 63, wherein the imaging chamber width is uniform and is between 0.5 mm and 2.5 mm.

82. 64. The method of claim 63, wherein the imaging chamber has a uniform depth and width, the width being between 10 and 200 μm.

83. 64. The method of claim 63, wherein the imaging chamber has a uniform depth and a serpentine shape in plan view.

84. 84. The method of claim 83, wherein an outer turning diameter of the serpentine imaging chamber is about twice an inner turning diameter of the serpentine imaging chamber.

85. 64. The method of claim 63, wherein the imaging chamber has a uniform depth and the shape of the imaging chamber in plan view is serpentine in shape, having a width of 1.25 mm, an inner turn diameter of 1.25 mm, an outer turn diameter of 2.5 mm, and a depth of 0.125 mm.

86. 64. The method of claim 63, wherein the imaging chamber has a helical shape in plan view.

87. 64. The method of claim 63, wherein the imaging chamber has a contour in plan view that is irregular like a chest wall.

88. 64. The method of claim 63, wherein the imaging chamber has a geometry such that a distribution of particles in the mixture remains substantially homogenous when the mixture is transferred into the imaging chamber.

89. 64. The method of claim 63, wherein the step of mixing comprises mixing the sample with a diluent and a stain.

90. 90. The method of claim 89, wherein the step of mixing comprises mixing a stain in dry form.

91. 90. The method of claim 89, wherein the step of mixing includes mixing with a liquid stain.

92. 90. The method of claim 89, wherein the known sample amount is between 0.1 μL and 10 μL and the volume of diluent and stain is between 10 μL and 500 μL.

93. 64. The method of claim 63, wherein the step of mixing comprises mixing the sample with a diluent, a staining agent and a sphering agent.

94. 64. The method of claim 63, wherein the steps of separating, mixing, transferring, acquiring, counting, and calculating are applied to a sample comprising at least one of blood cells, platelets, sperm, bacteria, spores, and inorganic particles.

95. 1. A single-use cartridge for use with an apparatus, including an automated microscope, for analyzing blood cells in a biological sample, comprising: a) a sample collection port for receiving the sample; b) a sample metering valve for isolating a predetermined volume of said sample; d) a mixing chamber in communication with the sample metering mechanism and in communication with a diluent source for mixing the separated volume of the sample with the diluent; e) an imaging chamber in communication with said mixing chamber and adapted to enable acquisition of images of said blood cells from said biological sample by said automated microscope.

96. 96. The cartridge of claim 95, wherein the imaging chamber has a volume sufficient to accommodate all of the predetermined volume of sample from the metering chamber when mixed with an unknown portion of the liquid reagent and has a depth such that the blood cells are substantially separated and do not overlap with images acquired by the automated microscope.

97. 96. The cartridge of claim 95, wherein the metering valve is part of a movable valve mechanism configured to (1) create fluid communication between the sample collection port and the metering chamber at a first position, and (2) create fluid communication between the sample contained in the metering chamber and the diluent channel, the imaging chamber, and the mixing chamber at a second position.

98. 98. The single-use cartridge of claim 97, wherein the movable valve mechanism is a rotary valve having a cylindrical axis.

99. 99. The single-use cartridge of claim 98, wherein the metering chamber is a through conduit within the cylindrical shaft.

100. 99. The single-use cartridge of claim 98, wherein the metering chamber is a recessed volume on a surface of the cylindrical shaft.

101. 96. The single-use cartridge of claim 95, wherein the liquid reagents are pre-packaged in containers on the cartridge.

102. 96. The single-use cartridge of claim 95, wherein the liquid reagent is contained in a container external to the cartridge, and the diluent channel is adapted to provide fluid communication between the cartridge and the external container.

103. 96. The single-use cartridge of claim 95, wherein the liquid reagent comprises a sphering agent, such as a zwitterionic surfactant, that isovolumically reshapes the red blood cells.

104. 96. The single-use cartridge of claim 95, wherein the liquid reagent comprises an antibody conjugated to a detectable label that targets a specific blood cell or a specific antigen associated with a blood cell.

105. 96. The single-use cartridge of claim 95, further comprising a photometric measurement chamber in fluid communication with the sample collection port.

106. 96. The single-use cartridge of claim 95, wherein a portion of the imaging chamber is a photometric measurement chamber.

107. A blood cell analyzer holding the cartridge described in claim 95 within the field of view of a microscope, the blood cell analyzer further comprising a processor responsive to the microscope and operative to count at least a portion of all of the particles within the imaging chamber of the cartridge.

108. 1. A single-use test cartridge for use with an apparatus, including an automated microscope, for analyzing blood cells in a diluted biological sample, comprising: a) an input port for receiving the mixed sample; b) an imaging chamber having a volume sufficient to contain all of the mixed sample, including a length at least 10 times its width and a depth at which the blood cells are substantially separated and do not overlap in an image acquired by the automated microscope; c) a cartridge comprising a vent in fluid communication with said input port and said imaging chamber.

109. 109. The single-use cartridge of claim 108, wherein the imaging chamber has a serpentine shape in plan view.

110. 110. The single-use cartridge of claim 109, wherein the imaging chamber has a total volume of less than 100 microliters and a depth of less than 200 μm.

111. A blood cell analyzer holding the cartridge described in claim 108 within the field of view of a microscope, the blood cell analyzer further comprising a processor responsive to the microscope and operative to count at least a portion of all of the particles within the imaging chamber of the cartridge.

112. 2. The subject matter of any one of the preceding claims, wherein the dilution ratio of sample to diluent is from 1:10 to 1:250.

Images