WO2001094938A1 - Flow cytometry-based hematology system - Google Patents
Flow cytometry-based hematology system Download PDFInfo
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- WO2001094938A1 WO2001094938A1 PCT/US2001/016814 US0116814W WO0194938A1 WO 2001094938 A1 WO2001094938 A1 WO 2001094938A1 US 0116814 W US0116814 W US 0116814W WO 0194938 A1 WO0194938 A1 WO 0194938A1
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- light
- particles
- flow cytometry
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- cells
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- H04L25/00—Baseband systems
- H04L25/02—Details ; arrangements for supplying electrical power along data transmission lines
- H04L25/06—Dc level restoring means; Bias distortion correction ; Decision circuits providing symbol by symbol detection
- H04L25/061—Dc level restoring means; Bias distortion correction ; Decision circuits providing symbol by symbol detection providing hard decisions only; arrangements for tracking or suppressing unwanted low frequency components, e.g. removal of dc offset
- H04L25/063—Setting decision thresholds using feedback techniques only
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
- G01N15/1459—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
- G01N35/1095—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1468—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle
- G01N15/147—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream
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- G01N2015/011—
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- G01N2015/014—
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- G01N2015/016—
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- G01N2015/018—
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1486—Counting the particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1493—Particle size
Definitions
- This invention relates in general to bioparticle analysis, and more specifically to methods and devices for performing automated blood cell analysis by integrating unconventional flow cytometry light scatter techniques with a unique approach to reagent delivery and sample processing that minimizes reagent waste. More particularly, the present invention is directed to a flow cytometry-based blood sample analysis system that employs a set of selected features which, in combination, provide at least a complete blood count, a five-part differential white blood cell count, and a reticulocyte count in a system that is small, reliable, and inexpensive enough to be well suited for use in small clinical laboratories, doctor's offices, veterinarian offices, and the like. The system may also be used in the analysis of any biological sample other than blood (e.g., urine) that is amenable to analysis by standard flow cytometry techniques. The present invention also relates to methods for use of the flow cytometry-based blood sample analysis system.
- Mammalian peripheral blood usually contains three major classifications of blood cell types - red blood cells (“RBCs”), white blood cells (“WBCs”), and platelets ("PLT”). These cells are suspended in a solution referred to as plasma, which contains many different proteins, enzymes, and ions.
- the functions of the plasma components encompass blood coagulation, osmolality maintenance, immune surveillance, and a multitude of other functions.
- RBCs are responsible for oxygen and carbon dioxide transport within the circulatory system.
- normal mature red cells have a bi-concave cross- sectional shape and lack nuclei.
- RBCs can range in diameter between 4 and 9 microns, depending on the species, and have a thickness that is generally less than 2 microns.
- hemoglobin a protein known as hemoglobin, which performs the dual roles of oxygen and carbon dioxide transport and which is present at a very high concentration.
- Hemoglobin is responsible for the overall red color of blood, due to the presence of iron in the molecule.
- erythrocytes red blood cells
- red cells red cells
- RBCs red blood cells
- RBCs nucleated red blood cells
- a reticulocyte is a red blood cell precursor that has completed most of the normal cell development stages in bone marrow, and has expelled its nucleus. The last portion remaining to leave the reticulocyte before it becomes a truly mature RBC is transfer RNA.
- the detection of reticulocytes is important in clinical evaluation of a patient's ability to produce new red blood cells.
- the reticulocyte count also can be used to distinguish among different types of anemia. In anemia, red cell production is diminished to the point where it can no longer keep up with red cell removal, and as a result the overall red blood cell count and hematocrit are low.
- the presence of an increased number of reticulocytes in these patients provides evidence that the bone marrow is functioning, and attempting to make up for the red blood cell deficit.
- WBCs White blood cells
- RBCs red blood cells
- white blood cells are used interchangeably to refer to the non-hemoglobin-containing nucleated blood cells present in the circulation as described above.
- Measurements of the numbers of white blood cells is important in the detection and monitoring of a variety of physiological disorders. Elevated numbers of abnormal white blood cells are a sign of leukemia, which is an uncontrolled proliferation of a myelogenous or a lymphogenous cell. Neutrophilia, or greatly increased numbers of neutrophils, is an indication of inflammation or tissue destruction in the body, by whatever cause.
- White blood cells may be broadly classified as either granular or agranular. Granular cells, or granulocytes, are further subdivided into neutrophils, eosinophils, and basophils. Agranular white cells are sometimes referred to as mononuclear cells, and are sub-classified into lymphocytes and monocytes.
- Measurements of these two major WBC sub-classifications make up what is referred to as a two-part WBC differential count (or two-part differential).
- Measurements of the components of these subclassifications neurotrophils, eosinophils, basophils, lymphocytes, and monocytes, produce a five-part WBC differential count (or five-part differential).
- Neutrophils are the most prevalent of the five major classes of white cells, usually making up a little over half of the total number of white blood cells. Neutrophils are so named because, they contain granules within their cytoplasm, which can be stained with a neutral pH stain. These cells have a relatively short life span, on the order of a day or less. Neutrophils attack and destroy invading bacteria, and other foreign agents in the tissues or circulating blood as part of the body's immune response mechanisms. Eosinophils are the next most prevalent of the granulocytes, behind the neutrophils, but generally account for less than five percent of the total number of white blood cells.
- Eosinophils also contain granules within their cytoplasm, which can be stained with an eosin stain. Like neutrophils, these cells do not exist for long periods of time in the peripheral blood. Eosinophils play a part in the body's immune response mechanisms that are usually associated with allergies or parasitic infections.
- Basophils are the least common of the granulocytes, and the least common of all the five classifications of WBCs. As they are granulocytes, they contain granules within their cytoplasm which can be stained, in this case using a basic (high pH) stain. These cells also are known to play a role in the body's immune response mechanisms, but the specifics are not certain.
- Lymphocytes are the most prevalent of the mononuclear cell types, and generally make up between 20 and 30 percent of the total number of white blood cells. Lymphocytes have no granules in their cytoplasm, and their nucleus occupies a large majority of the cell volume. The thin area of cytoplasm of lymphocytes can be stained, as it contains RNA. Lymphocytes circulate in the peripheral blood, leave the circulation as part of the body's immune response mechanism, and eventually return to the peripheral blood. Many lymphocytes are relatively long-lived cells. Monocytes are immature forms of macrophages that, in themselves, have little ability to fight infectious agents in the circulating blood.
- Normal animals will generally have between 1-5x10 11 platelets per liter. These cellular particles are usually much smaller than RBCs, having a diameter between 1 and 3 ⁇ m. Platelets are formed as buds from the surfaces of megakarocytes, very large cells found in the bone marrow that do not themselves leave the marrow to enter the blood circulation. Rather, the buds pinch off and enter the circulation as platelets. Like RBCs, platelets lack nuclei and thus cannot reproduce. Functionally, platelets act to aggregate so as to plug or repair small holes in blood vessels. In the case of larger holes, platelet aggregation acts as an early step in clot formation. As a result, platelet count and function are clinically very important. Abnormally low platelet counts may be the cause of a clotting disorder.
- CBC complete blood count
- CBC a CBC
- 5-part differential and a reticulocyte count are a common diagnostic procedure performed to diagnose, track and treat an abundance of ailments.
- These tests make up the great majority of hematology analyses that are performed in human and animal clinical laboratories around the world. These three tests can all be performed using a microscope, centrifuge, counting chamber, slide, and appropriate reagents.
- the skills necessary to perform these test manually are rare and take years of training.
- the time required to perform each of these tests manually is very high. As a result, significant automation via instrumentation has been pursued in this field since the early 1950's, when the first Coulter impedance cell counters appeared.
- An impedance counter consists of two chambers filled with a saline solution and connected via a small orifice. In the presence of a constant voltage across the orifice, cells passed individually through the orifice displace a volume of saline solution and thereby increase the impedance of the orifice.
- the size of the cell can be related to the voltage pulse generated by passage of the cell through the orifice. As a result, when a cell passes through the orifice, its presence and size can be determined from the resulting voltage pulse.
- impedance technology evolved, automated cell counters were developed that were able to count RBCs, WBCs, and platelets simultaneously on the same instrument, based on the different sizes of these classes of cells.
- Flow cytometry is a powerful method of analysis to determine the cellular content of various types of samples, and in particular samples that contain living cells.
- flow cytometers are useful for lymphocyte counting and classification, for immuno logical characterization of leukemias and lymphomas, and for cross-matching tissues for transplants.
- cells in a fluid solution are caused to flow individually through a light beam, usually produced by a laser light source. As light strikes each cell, the light is scattered and the resulting scattered light is analyzed to determine the type of cell.
- the cell may also optionally be labeled with a marker linked to a fluorescent molecule, which fluoresces when light strikes it and thereby reveals the presence of the marker on the cell.
- fluorescent molecules include FITC (fluorescein isothiocyanate), TRITC (tetramethyl rhodamine isothiocyanate), Texas Red (sulforhodamine 101), and PE (phycoerythrin).
- intracellular components of the cell such as nucleic acids, may be stained, and subsequently detected by fluorescence.
- fluorescent compounds include ethidium bromide, propidium iodide, YOYO-1, YOYO-3, TOTO-1, TOTO-3, BO-PRO-1, YO-PRO-1, and TO-PRO-1.
- Blood cell measurements made using flow cytometry often employ two separate measurements - one to measure the RBCs and platelets, and the other to measure the much less common WBCs.
- the reason for separate measurements is that the RBCs are much more common than other blood cell types, and thus detection of other cell type in the presence of RBCs requires that the RBCs either be removed or large volumes of sample be measured.
- these cells may be distinguished on the basis of immunochemical staining of particular cell surface antigens and/or differential cell type staining.
- U.S. Patent No. 5,047,321 (Loken et al.)
- a single step method for the analysis of blood cells employs at least two fluorescent dyes and a labeled cell marker to differentiate among the cells.
- the method still requires large volumes of sample to provide sufficient statistical data with regard to the less common blood components.
- Light scattering measurements are widely used in flow cytometry to measure cell sizes and to distinguish among several different types of cells. It is known that incident light is scattered by cells at small angles (approximately 0.5 - 20°) from the line traveled by the incident light that interrogates the cells, and that the intensity of the scattered light is proportional to the cell volume. The light scattered at small angles is referred to forward scattered light. Forward scattered light (also called forward light scatter, or small- angle scatter for angles of scatter between 0.5 - 2.0°) is useful in determining cell size. The ability to measure cell sizes is influenced by the wavelength employed and the precise range of angles over which light is collected.
- material within the cells having a strong absorption at the illuminating wavelength may interfere with the size determination because cells containing this material produce smaller forward scatter signals than would otherwise be expected, leading to underestimates of cell size.
- differences in refractive index between the cells and the surrounding medium may also influence the small-angle scatter measurements.
- Different cell types may be distinguished on the basis of the amount of orthogonal light scatter (or right angle side scatter) they produce.
- Cells having a high degree of granularity such as blood granulocytes, scatter incident light at high angles much more than cells with low granularity, such as lymphocytes.
- forward and right angle side scatter measurements are commonly used to distinguish among different types of blood cells, such as red blood cells, lymphocytes, monocytes, and granulocytes.
- eosinophils may be distinguished from neutrophils and other lymphocytes and other granulocytes on the basis of polarization measurement of right angle side scatter. Normally, incident polarized light is scattered orthogonally and remains polarized.
- U.S. Patent No. 5,492,833 discloses a method for distinguishing erythrocytes from reticulocytes by treating the cells with a ghosting reagent, followed by measuring light scatter.
- U.S. Patent No. 5,559,037 discloses a method for counting nucleated red blood cells (NRBCs) by first lying red blood cells, then exposing the NRBCs to a nuclear stain and measuring fluorescence and light scatter.
- NRBCs nucleated red blood cells
- 5,733,784 (Studho ne et al.) teaches a method for measuring the reticulocyte concentration in a blood sample that employs a reticulocyte staining reagent that is incubated for a period between 15 minutes and 4 hours before light scatter measurements are taken.
- U.S. Patent No. 5,879,900 discloses a method for simultaneous and quantitative analysis of damaged WBCs, NRBCs, and WBC subpopulations that employs multi-dimensional light scatter and fluorescence measurements.
- 5,891,734 discloses a system for differentiating blood cells in a blood sample that aspirates a portion of blood and mixes it with a fluorescent reagent, then automatically runs the stained sample through a flow cytometer and measures multi-angle light scatter and fluorescence.
- the samples must be standardized with respect to the amount of light scatter, fluorescence or impedance associated with standardized populations of the cells.
- the flow cytometry instrument itself must be calibrated to ensure proper performance. Calibration of the instrument is typically accomplished by passing standard particles through the instrument, and measuring the resulting scatter, fluorescence, or impedance. Flow cytometers may be calibrated with either synthetic standard materials (e.g., polystyrene beads) or with cells or other biological material (e.g., pollen or stained nuclei).
- US Patent No. 5,627,037 discloses a one-step method for determining absolute numbers of one or more populations of reticulocytes and/or leukocytes in a whole blood sample, using flow cytometry.
- the method comprises mixing a blood sample with a diluent, wherein the diluent contained a fixative, one or more fluorescent cell markers and a known number of fluorescent microparticles per unit volume, and thereafter analyzing the sample by flow cytometry.
- the fluorescence due to the microparticles must be distinguishable from that due to the cell markers.
- the flow cytometer is run with a fluorescence trigger set such that all the cells and the microparticles exceed the trigger level.
- the events then are reanalyzed under conditions where the microparticles are distinguishable from the cell markers.
- the number of microparticles and the number of cells are then counted.
- the number of cells and the number of microparticles provides a ratio that can be used, knowing the number of microparticles per unit volume and the total sample volume, to calculate the absolute number of cells in the whole blood sample.
- the method described in US Patent No. 5,627,037 relies on two analytical steps to provide the absolute number of cells per sample, and does not teach or suggest that the flow cytometer could be simultaneously standardized using the microparticles included in the sample.
- 5,380,663 discloses a method for calibrating a flow cytometer, which employs combined populations of fluorescent microbeads and a software program matched to each combined population of microbeads.
- Each microbead batch contains at least two different populations of fluorescent microbeads, and each population has a different fluorescence intensity.
- the software program contains information on the fluorescence intensity of each population of microbeads within the batch.
- the software program monitors the fluorescent microbeads for fluorescence degradation, and alerts the operator when the fluorescence intensity falls below acceptable levels.
- the method does not provide a means to automatically standardize samples so that variability in measurements due to differences among flow cytometers is reduced or eliminated.
- US Patent No. 5,451,525 discloses a method for determining the total number of cells in a sample, that does not require a known sample volume.
- the method employs a known number of discrete particles which are added to a blood specimen before the specimen is assayed in a flow cytometer. By counting the added particles and the cells, and relating the number of counted particles to the number of added particles, the number of cells in the entire blood sample can then be determined without need to measure the volume of the sample measured. Because the volume of the sample measured is unknown, the method disclosed is incapable of determining whether the proper sample dilutions have been made. The method further does not disclose an automatic means to calibrate the instrument to ensure that the variability in measurements due to differences among flow cytometers is reduced or eliminated.
- high- end hematology systems are designed to be high-throughput machines, so often multiple different specimen samples are being processed simultaneously, requiring different combinations of treatments and analyses.
- Analysis of red blood cells and white blood cells require different reagent systems, and are often optimized in a way that requires completely different hydraulic paths.
- To simultaneously perform both red blood cell and white blood cell analyses completely separate and distinct hydraulic paths are created for each type of sample.
- This combination of multiple simultaneous sample hydraulic paths creates undesirably high complexity, due to the use of large numbers of valves, vacuum lines, reaction chambers, syringes, and the like.
- CBC complete hematology assessment
- reticulocyte count a complete hematology assessment
- Instrumentation such as impedance counters that perform CBC with limited WBC differentials, are available for these in-clinic laboratories, but systems that can produce five-part differentials and reticulocyte counts are not.
- manual methods are employed. These manual methods, however, are slow, labor-intensive, and prone to operator error.
- the low-end impedance counters available to the clinics and doctor's offices includes such systems as the Abbott 1200, ABX Micros, and the Sysmex K-21. While the high-end hematology systems improved by incorporating flow cytometric techniques and other advances, the technology used in the low-end systems has changed very little. These low-end units have become much more compact and efficient through incorporation of semiconductor microelectronics where applicable; however, in the end these systems are functionally no different from the Coulter instruments used by clinical laboratories in the late 1970's and early 1980's. In particular, these instruments still cannot perform a five- part WBC differential, nor can they produce a reticulocyte count.
- the QBC ® system does not utilize any liquid reagents, and is therefore very reliable.
- This method is also a unit dose method, in that a single capillary tube and float are used for each analysis. As a result, there is no wasted reagent.
- a laser diode can also reduce the cost of the laser system.
- laser diodes do not have the optical characteristics of gas lasers. They are astigmatic, and have temperature-dependent noise regions, which are referred to as "mode-hopping". This mode-hopping noise makes detection of small particles such as platelets very difficult. Controlling the temperature of the laser diode to a quiet thermal area can reduce this mode-hopping problem. This can be accomplished by a variety of temperature feedback systems, such as a thermal electric cooler, which keeps the temperature of the diode laser in a very narrow temperature range.
- any type of thermal control is costly, especially since the laser diode itself is a source of heat. Thus even the act of turning on the diode laser causes mode-hopping effects until thermal equilibrium is reached, which could take five to ten minutes.
- Other than the laser light source there is significant costs and complexity associated with beam shaping, and light collection optics for flow cytometry type instruments.
- Beam shaping optics are generally intended to take a circular gausian laser beam, and create an ellipse where the power distribution is both more intense and more evenly distributed over the area of interest.
- Many hematology instruments use a collimated approach, with the tails of the gaussian curve being blocked by an aperture. This creates a top hat effect, which gives uniformity over the region of interest, and reduces the need for fluidic control, and laser pointing stability, but sacrifices power density at the cell.
- the traditional approach has been to first expand the initial laser beam, so that it may then be focused to a small spot size.
- Both the expansion step and the focusing step are accomplished using various optical elements, such as lenses, beamsplitters, and mirrors.
- optical elements such as lenses, beamsplitters, and mirrors.
- significant costs are incurred by having many optical components.
- each optical component incurs further costs in that each one needs to be held securely in space, and carefully aligned.
- Light collection optics in flow cytometer systems are even more complex than the ' beam shaping optics. This is because in almost all applications, more than one measurement of light is made. To accomplish this, two or more detection sources are needed. Each detector requires several optic components that steer light from the area of interest to the detector. As in the beam shaping case, significant costs are incurred by the requirement for numerous optical components, each of which needs to be held in space and carefully aligned.
- This device is a flow cytometry based hematology system that has high level of reliability, and utilizes very low volumes of method reagents to determine at least a complete blood count (CBC), a five-part differential white blood cell count, and a reticulocyte count.
- CBC complete blood count
- a five-part differential white blood cell count a reticulocyte count.
- the system of the present invention employs a selected set of features that, in combination, create a device that is capable of producing a complete hematology analysis, but which does not have the drawbacks of the prior art.
- the system is a flow cytometer that includes a lensless optical detection system for collection of light emitted from a laser light source, after interacting with a cell or particle.
- the laser light source is a standard diode laser light source that is shaped to produce a thin line, as disclosed in copending U.S. Patent Application No. 09/********.
- the shape of the laser light beam is well-suited for the detection of scattered light.
- the laser light further is Furthermore, the reliability of the system is further enhanced by use of a photodetector array that does not include any moving parts, nor require any lenses.
- the photodetector array is such that the scattered light is detectable, and this data collected provides a complete hamato logical analysis, when used in combination with a high numerical aperture technique.
- the system uses a unique system of consumable reagent tubes that act as reaction chambers, mixing chambers, and waste chambers for the blood sample analyses.
- the consumable tubes incorporate latex particles or the like (fixed cells), which act as internal standards to ensure that the dilutions made during processing of the samples have been carried out correctly. The use of these consumable tubes results in less waste and greatly improved accuracy and validation of the results.
- one object of the present invention is to provide a lensless light collection system for a flow cytometer.
- a diode laser the diode laser producing light to irradiate cells in the flow cell
- a lensless optical module the lenless optical module having a first portion capable of collecting light scattered at two or more forward scatter angle ranges, and a second portion capable of collecting light scattered at a high numerical aperture
- a signal processor the signal processor being capable of analyzing one or more signals generated from the first portion and the second portion of the lensless optical module, and generating data therefrom; and a data processor for analyzing the data.
- FIG 1 is a schematic representation of the system hydraulics, which shows the systems valves (1,2,3,4,5), syringes (6,7), bulk reagent bottles (8,9), and other major components of the system.
- FIG 2 is a drawing of the flow cell assembly used in the optical detection system. This contains three tubing ports (20, 21, 23) which allow laminar flow through the quartz flow channel (22) where the laser interacts with the cells.
- FIG 3 A and FIG 3 B are schematic representations of the laser beam shaping optics.
- Figure 3 A give a top view representation
- figure 3 B gives a side view representation.
- the raw laser diode, 30, emits light that is diverging in two separate axis's.
- a collimating lens, 31, eliminates the divergence in both axis's, and illuminates lens 32.
- Lens 32 acts to converge the beam in one axis such that nearly all of the light from the laser beam may pass through the width of the flow channel of 22. W represents this width.
- a third lens, 33 is introduced between 32 and W, such that it produces a sharp focus at W.
- the focal point of lens 33 is the desired intersection of laser light, and the particle or cell of interest.
- FIG 4 is an illustration of the cell detection system, which shows cells or particles flowing through 22, which is held in space around at least two, but preferably four photodiode detectors, without any collection or imaging lenses. These detectors collect orthogonal scatter, 42, low angle forward scatter, 45, axial light loss or extinction, 44, and high angle forward scatter, 43. Laser light is illuminated on 22 via 41, which contains the beam shaping optics of figure 3.
- FIG 5 is a drawing of the photodiode mask which accommodate three independent optical measurements in the forward direction of the laser.
- Low angle forward scatter is masked by 51 , axial light loss by 52, and high angle forward scatter by 53.
- the preferred embodiment these three area are on a single fabrication 50,of semiconductor material where only the desired geometry is an active part of the semiconductor material.
- Other approaches would have 50 being a metal mask that could sit over three discrete photodiodes.
- FIG 6 is a schematic representation of what happens in the consumable during the system cycle.
- the consumable tube, 60 starts with a small amount of method reagent, to which precisely metered amounts of whole blood via 6, or other unknown material is added with the systems sheath fluid, from 8, via 7, though the hydraulic needle 11. This creates 60, which is then analyzed through the optics of Figure 4.
- To create 62 more whole blood or other unknown material is added to the consumable tube.
- a lytic agent and sheath fluid are added to create
- FIG 7 is drawing of the tube positioning assembly that moves the tubes shown in Fig 6.
- Slot A is intended for the patient sample of whole blood, or other type of appropriate specimen.
- Slot B is intended for the consumable tube depicted in Figure 6.
- Slot C is for a tube containing lyse that stays in aboard the system in slot C, for numerous runs of consumables (one per kit of greater than 20, ideally 50 consumables).
- Slot D is for an on board cleaner tube, that is also intended to act a primer tube, to properly position of fluids at system start up.
- FIG 8 is a drawing of the mixing and piercing assembly.
- the tube slots of Figure 7 are moved between a piercing station where the needles 11 and 12 are simultaneously inserted into stoppered test tubes by means of a motor 81.
- needle 12 vents the pressure change to an air filter 13, which is at atmospheric pressure.
- the carriage of Figure 7 moves the consumable under 80, where by through the use of several motors, including 82, the consumable is mix, making the solution inside it homogenous.
- FIG 9 is a drawing of the preferred embodiment of Figure 1.
- a manifold block holds valves that have appropriate hydraulic paths, and integrates the syringes and the motor drive mechanic.
- FIG 10 displays red cell (C), platelet (A) and latex (B) histogram data obtained from an embodiment of the system.
- Figure 10 A depicts histogram data of the digitized forward scatter high channel, 43, on the x-axis (0 through 1023, or 8 bits) verses the number of events (or occurrences) collected for each of the individual channels.
- Figure 10 B shows the same data run, except it depicts the extinction or axial light loss channel , 44.
- Figure 10 C shows the same data run, except it depicts the right angle scatter channel., 42.
- Figure 10 D shows the same data run, except it depicts the time of flight, or pulse width measurement. Pulse width measurements can be made off of any of the other channels that utilize a photodetector, but it is preferred to use either extinction, 44, or forward scatter low, 45.
- FIG 11 displays the same red cell, platelet and latex data from Figure 10, except in scatter-plot form.
- Scatter plots are simply a representation of data that is collected simultaneously from a cell or particle, from one measurement, plotted on the x-axis, against an other measurement plotted on the y-axis. Latex particles are distinguishable in A in Figure 11 A, and in B in figure 11 B.
- FIG 12 A displays white cell histogram data for the extinction channel which clearly show three populations, A are the lymphocytes, B are the monocytes and C are the granulocytes.
- Figure 12 B shows the same patient sample, except this run of data incorporated latex particles. Again, A are the lymphocytes, B are the monocytes and C are the granulocytes. The latex particles are indicated by D, and are easily distinguished from the white cells.
- Figure 12 C and 12 E show the same patient sample for the parameter of forward scatter low 12 C, and right angle scatter 12 E, without latex particles added.
- Figures 12 D and 12 F show the same patient sample for the parameters of forward scatter low 12 D, and right angle scatter 12 F, with latex particles added.
- FIG 13 A and 13 B displays the same white cell and latex data from figure 12 B, 12 D and 12 F, except in scatter-plot form.
- FIG 14 displays red cell and reticulocyte histogram data for right angle scatter,
- Figure 14 A and for extinction, Figure 14 B obtained from an embodiment of the system.
- FIG 15 displays red cell and reticulocyte scatter-plot data obtained from the same sample as depicted in figures 14 A and 14 B.
- the extinction (y-axis) and the right angle scatter (x-axis) show the separation of reticulocytes for the main population of mature red blood cells.
- the value of 1.72 % reticulocytes is in excellent agreement with the manual reference method of 1.60 %.
- FIG 16 displays white cell differential histogram data obtained from right angle scatter, Figure 16 A, and for time of flight, or pulse width, figure 16 B, from an embodiment of the system.
- FIG 17 displays white cell differential scatter-plot data obtained from the same sample as depicted in figure 16 A and figure 16 B.
- the extinction (y-axis) and the right angle scatter (x-axis) show the separation of several parts of the white cell differential.
- the area indicated by a represents lymphocytes
- the area indicated by B represents monocytes
- the area indicated by C represents neutrophil (a sub-set of granulocytes)
- the area indicated by D represents eosinaphils (another sub-set of granulocytes).
- the eosinaphil cells present in D account for 20.63 % of the total, which is in excellent agreement with a reference method of 21.70 % eosinaphils.
- FIG 18 displays a spectrophotometer scan of the reticulocyte dye solution, which would be representative of 61, without the presence of blood.
- FIG 19 A displays a spectrophotometer scan of lyse a whole blood solution, which is read at 540 nm for a hemoglobin reference method (cyan-hemoglobin).
- Figure 19 B displays a spectrophotometer scan of lyse a whole blood solution, which when read at 540 nm for a hemoglobin, indicates the oxy-hemoglobin, and at 580 represents the deoxy-hemoglobin..
- FIG 20 displays a spectrophotometer scan of diluted whole blood solution.
- FIG 21 displays a spectrophotometer scan of a lyse whole blood and reticulocyte dye solution.
- FIG 22 displays a spectrophotometer scan of a diluted whole blood and reticulocyte dye solution.
- FIG 23 displays the spectrum analyzer output of a diode laser operating in single mode Figure 23 A, and at the same laser operating with high frequency modulation, Figure 23 B.
- the peaks in Figure 23 A show high levels of noise at many different frequencies. While the baseline for this is excellent the high frequency noise peaks cause significant problems when trying to implement an extinction measurement and a lens-less scatter collection system. The net effect is false event counting and improper quantification of pulse height or area.
- the spectrum of the high frequency modulated laser in Figure 23 B does not have as low of a baseline level, but has no high spikes. This helps enables accurate extinction quantification, and the implementation of a lens-less collection system for the other channels.
- FIG 24 displays a plot of noise (root mean square) verse temperature for a constant power driven laser diode, or single mode, and the same device driven with high frequency modulation. This shows that a single mode laser that is driven at a constant power, can achieve noise levels at or below that of high frequency modulated lasers in some temperature ranges.
- high frequency modulation or a temperature-controlled laser is capable of enabling accurate extinction quantification, and the implementation of a lens-less collection system for the other channels. Because of the cost of controlling temperature, is much greater than the cost of oscillating (modulating) a laser, the preferred embodiment is high frequency modulation.
- FIG 25 is a block diagram of the system electronics.
- FIG 26 is a block diagram of the signal processing electronics.
- the present invention discloses a flow cytometry-based hematology system that is capable of determining at least a complete blood count (CBC), a five-part white blood cell differential, and a reticulocyte count.
- CBC complete blood count
- the system has high level of reliability, and utilizes very low volumes of method reagents.
- the system is very inexpensive to construct, and further is usable without the need for significant levels of operator training.
- the system is particularly well suited for use in environments where there is a need for an instrument that can analyze large numbers of blood samples quickly and inexpensively and provide a complete blood analysis for each sample. Such environments would include, for example, medical clinics, doctor's offices, and veterinary offices.
- the flow cytometry-based hematology system of the present invention combines a unique lensless optical detection system with the use of consumable tubes containing reference particles that are used for optical standardization and to check if the instrument is performing dilutions correctly.
- the system also employs a diode laser having a certain beam shape that is critical for the successful operation of the system.
- the use of a diode laser for bioparticle analysis in flow-cytometry-based systems has heretofore largely been avoided, due to the propensity of these systems to exhibit periodic noise phenomena.
- the present inventors have discovered that such problems can be overcome through the use of a high frequency modulation technique, discussed in greater detail below.
- CBC complete blood count
- reticulocyte count a system that is much lower in cost and higher in reliability that existing flow-cytometry-based hematology systems, and which can measure at least a complete blood count (CBC), a five-part differential white blood cell count, and a reticulocyte count, without the need for fluorescence or light absorption measurements.
- CBC complete blood count
- reticulocyte count a system that is much lower in cost and higher in reliability that existing flow-cytometry-based hematology systems, and which can measure at least a complete blood count (CBC), a five-part differential white blood cell count, and a reticulocyte count, without the need for fluorescence or light absorption measurements.
- the system of the present invention is not precluded from incorporating fluorescence or light absorption measurements to measure one or more desired parameters, as described herein.
- the first item to be considered as part of the flow cytometry-based hematology system of the present invention is the consumable tube.
- Each consumable tube has a unique coded label, as does every other tube placed in the instrument.
- the coded label is a bar-coded label.
- the consumable tube contains one or more distinct types of particles that are used for count quantification checks, as well as for optical standardization.
- Each lot of reagents is aliquoted into consumable tubes, along with a defined number of reference particles.
- reference particles are known to those in the art and may be, for example, pollen grains, fixed cells or latex particles.
- Latex particles have a different index of refraction than cellular components, and many sizes of latex particles are commercially available. In the case of 4.1 -micron particles, these are distinguished from red blood cells and platelets on extinction (EXT), low angle forward light scatter (FSL), and right angle scatter (RAS) channels, but overlap with red blood cells on the high angle forward light scatter (FSH) and time-of-flight (TOF) channels. Functionally, it does not matter which axis has the best separation of the reference particle(s). As long as at least one of the five channels shows separation, the entire population of particles can be gated and analyzed completely separately from any of the sample cellular components.
- EXT extinction
- FSL low angle forward light scatter
- RAS right angle scatter
- FSH high angle forward light scatter
- TOF time-of-flight
- Fixed red blood cells from a variety of different species are often employed as reference particles. These cells may be completely hidden on all channels during red blood cell analysis, and therefore may not be appropriate for use in detection and counting of red blood cells. However, they are still usable in the detection and counting of white blood cells, because since the fixation process makes them immune to lysing systems, they would be easily distinguished on all channels during white cell analysis.
- Fixed red blood cells from avian species offer the added feature of having a nucleus, which helps distinguish them from mammalian red cells exclusively by right angle scatter.
- the internal reference particles are to be used at known concentration, and are to act as a cellular surrogate.
- This surrogate must have properties such that the instrument in at least one measurement technique can uniquely distinguish it from the cellular constituents of the sample.
- This cellular surrogate acts as an intra-sample, internal standard for quality control purposes. This assures that the instrument's measurements are performing properly, and that all dilutions have been made properly.
- the preferred embodiment of this surrogate is polystyrene particles. However, other particles, such as pollen, glass, fixed cells or large colloids could be used, so long as the above conditions are fulfilled.
- the particle size range is preferably from about 1 micron to about 10 microns, and have optical characteristics that uniquely separate them from platelets, white cells and red cells on at least one measurement axis of the instrument.
- Polystyrene particles are commercially available through several manufacturers, such as Seradyn and Duke.
- the preferred size is 4.0 microns, which is easily distinguished from the cellular components of blood along several axes of data collection in the instrument. Choosing this size for the particles allow the particle's concentration to be easily determined.
- the reagent systems within the consumable are sensitive to a percent solids measurement (volume of particle time concentration of particles); thus, the lower the volume chosen, the higher the concentration of particles, and the broader the range that may be practiced.
- the preferred embodiment is to have the concentration of particles in the consumable at 10,000 per ⁇ l. At this level, the number of reference particle events counted will be within the same range as the number of white blood cells counted.
- the concentration of the particles and their optical characteristics is determined by a reference method. Characteristics of the particles, such as counts per micro liter for each particle type, mean signal for an optical measurement for each particle type, and coefficient of variation for an optical measurement for each particle type, are obtained.
- Characteristics of the particles such as counts per micro liter for each particle type, mean signal for an optical measurement for each particle type, and coefficient of variation for an optical measurement for each particle type, are obtained.
- One of skill in the art would appreciate that other parameters associated with each particle type could also be measured and used advantageously in the system of the present invention, as desired, and the present invention is not intended to be limited in any way to the specific parameters listed above.
- an encrypted bar code label is generated that references any one of or any combination of at least the following parameters:
- the table of reference values may contain, as parameters, one or more of the following:
- the dye fluid will be tested for absorption at one or more wavelengths characteristic of these spectra, i.e., a wavelength at which absorption of the dye is at a maximal or minimal point, or which is uniquely identified with the dye.
- a reagent containing a dye may be tested for absorption at the following wavelengths:
- Measurement at 635 nm is useful to detect blue dyes, which absorb light at this wavelength. Hemoglobin absorbs green light strongly, so a measurement at 540 nm is used to measure hemoglobin concentration. Measurements at 580 nm are useful to detect the products of some clinical chemistry assays, which absorb yellow light. Finally, a measurement at 488 nm is useful as a true calibration wavelength, since neither hemoglobin nor any of the commonly used reticulocyte dyes absorb at this wavelength and thus minimally interfere with this measurement. As an example, assume new methylene blue is designated dye 3, and has the following absorption values in the reference table:
- the fourth lot of tubes for CBC tests contains two different sets of reference particles.
- the first set of reference particles references table entry 7, which refers to 4.1 -micron particles at a concentration of 1,200 particles per microliter (0712).
- the second set of reference particles references table entry 25, which refers to a fixed cell particle at a concentration of 32,500 per microliter (25325), in a new methylene blue solution, which is table entry D (d).
- the consumable tubes containing the above-described solution are serialized from 0 - 50,000.
- the system can also reference the expected optical values in the tables, which are independent of dilution. Comparisons of the data of the means and coefficient of variation (CV) of the particles, as they compare to the reference tables, allows a determination of whether a dilution error has occurred, and also gives insight as to optical alignment, laser power stability, and flow stream characteristics. As with the dilution example, if the recovered light scatter data does not match expected data within a preset tolerance, some or all of the cell classification analysis will not be reported. In this fashion, an accurate error message can be displayed to the operator that will assist in the proper remedial measures. Furthermore, the system can record its performance over time, and maintain a database of the system performance over time to record trends in the system. Based on the data obtained, and the recent trends, recommendations for service, recalibration, or troubleshooting techniques can be automatically generated.
- the ideal whole blood dilution is 1:100 for analysis of RBCs, which utilizes less than 20% of the volume of the consumable tube. This leaves 80%, or more of the tube volume to perform a WBC dilution, and a final rinse out of the system (waste chamber).
- the sensing zone must be further compressed from methods currently available. This can be accomplished in two ways. The first is to control the mass flow rate to very low, but consistent rates (0.05 microliters per second), to keep the core stream on the order of 7 microns wide. The second is to limit the laser beam height to around 3 microns. This was accomplished through the use of a diode-laser line-emitting system, as described in pending U.S.
- the apparatus contains a semiconductor laser that produces light output that is oriented in first and second mutually perpendicular axes that are both perpendicular to the direction of light propagation.
- the apparatus also includes an optical system that is arranged in a manner so as to focus the output of the laser in the first and second axes at first and second focal parts that are spaced apart in the direction of the laser light propagation. At a plane intersecting each of the focal points, the light is formed into a line having a width in one axis and a length in the other axis.
- Classical flow cytometers and flow cytometry-based hematology systems collect light that is scattered from cells at a variety of angles.
- detectors without any accompanying lenses or other optical elements, are fixed in space to collect the appropriate signals induced by the cell passing through the laser beam.
- These signals include extinction (EXT) (0°- -0.5°); low angle forward scatter (FSL) ( ⁇ 1°- -3°); high angle forward scatter (FSH) ( ⁇ 4° - -9°); and right angle scatter (RAS) ( ⁇ 50° - ⁇ 130°).
- EXT extinction
- FSL low angle forward scatter
- FSH high angle forward scatter
- RAS right angle scatter
- the first focus is the plane in which the laser light intersects the cores stream of cells / particles. This focus is perpendicular to the plane of travel of the cells, and light scatters due to passage of the cells orthogonally through the thin center (focal plane) of a wide beam.
- a beam with a minimal height i.e., a minimal dimension in the direction of particle flow through the flow celfh large numbers of cells, at high concentrations, can be analyzed without interference from coincidence events. This minimizes liquid volumes and the need to handle large volumes of liquids.
- this arrangement produces a high light power distribution on the cells at this first focus, so that right angle light scatter (RAS) can be measured using a much less expensive detector than that which is employed on conventional systems.
- RAS right angle light scatter
- a photodiode can replace the use of a photomultiplier tube.
- the horizontal power distribution allows for flow stream wander in that axis without appreciable drift, and further minimizes light scatter from the edges of the flow cell by maintaining the light power distribution parallel to the flow cell channel.
- the second focus is at the plane where the detectors are placed.
- the wide beam portion from the flow cell is reduced down to a minimum (i.e., is focused), and the light that has passed through the first focus has expanded, forming a long line of light having a certain height.
- a photodiode detector is placed at the point where the light level is greatest. This is the extinction (EXT) detector, which measures all of the axial light hat is lost (the sum of absorbed light and scattered light) as the cell crosses the laser beam. This measurement hold a great deal of information, but this information can only be extracted when there is a high signal to noise (S/N) ratio.
- the gas lasers conventionally employed in flow cytometer-based hematology systems produce strong signals, but high levels of noise associated with these lasers have minimized their utility for EXT measurements. This can be offset by utilizing longer plasma tubes, as the longer the plasma tube the lower the noise level, and thus the greater the S/N ratio.
- Diode lasers have inherently lower noise levels than gas lasers, but exhibit periodic high levels of noise. These intermittent bursts of noise are referred to as "mode hopping". Subtle temperature or electrical current changes cause the laser diode to abruptly change from one single mode state to another single mode state. When mode hops are occurring, the S/N ratio of the extinction measurement is very low, making it useless for measuring small particles, or to distinguish subtle difference in larger ones.
- each laser diode must be individually characterized to find a quiet temperature/current area where mode hops do not occur. Laborious effort must be incurred to characterize each individual- laser diode for the ideal temperature and current settings. In addition, as the laser ages, the quiet temperature/current area changes, and resetting the temperature and current controls. Furthermore, even if the proper temperature is determined, when a diode laser is first turned on, it generates its own heat. Temperature equilibrium of the diode laser can take up to 30 minutes to establish. This equilibration time decreases the useful life of the laser. Lastly, the implementation of temperature control severely limits the operating temperatures which a unit can be used, and adds significant cost and size to a relatively cheap and small laser diode.
- High frequency modulation can be employed.
- High-frequency modulation eliminates mode hops by having the laser exist in a multi-mode state.
- the primary mode of the diode changes, but since the diode laser is running in many modes to begin with, these changes do not produce any noise spikes.
- the frequency of modulation can chosen to be much, much greater than the frequencies of interest for cell events, so the modulation is invisible to the system.
- High frequency modulation solves the problems associated with use of temperature and current control in addressing mode hopping.
- a high frequency modulation system is stable within seconds, so there is no need to control for the temperature generated by the diode laser upon activation. Thus, the useful laser life is extended.
- high frequency modulation systems are stable over a large range of operating temperatures. With high frequency modulation, there is no need to find the quiet temperature/current area, and no need to reset the equipment as the laser ages. Thus, the labor cost incurred in finding and maintaining a quiet temperature/current area for the diode laser is avoided.
- High-frequency modulation is imposed on a diode laser in the following manner.
- a diode laser running in continuous wavelength (CW) mode has a built-in photodiode that is used for feedback, so that the power does not vary with temperature.
- This circuit changes the current input to the diode of the laser so that the light power is kept constant.
- the CW set point of the laser driver is set so that it produces one-half of the rated power output of the laser.
- a modulating circuit which uses a crystal oscillator to produce an electric current with a sine wave output, is "summed" with the CW current.
- the laser power output takes on a sine wave shape, with a power maximum near the maximum rated power level of the laser at the peak of the sine wave and almost zero power at the minimum of the sine wave.
- FSL low angle light scatter
- FSH forward light scatter
- the photodetector is preferably in the form of an photoarray on a single chip, with the detectors spaced and oriented to collect the scattered light at the appropriate angles.
- This configuration allows for three or more independent measurements to be made, without the requirement of any collection or imaging lenses. While the photodetector array has been described for the collection of EXT, FSL and FSH, such an array could be extended to angles of collection as high as -40°. Above this value, the channel of the square flow would interfere with measurements.
- a mask which acts as a light filter, can be place over the discrete diodes or the photodiode array.
- This mask preferably has openings with curved shapes, to preserve the true angles of light scatter by covering the corners of any square or rectangular photodiodes that are used..
- This masked set of photodiodes is preferably positioned such that the extinction sensor has the maximum level of incident light, i.e., at the second focus of the laser light beam.
- the position of the right angle scatter photodiode is essential to preserve a high numerical aperture, which can only be achieved without the use of a lens. Therefore, the photodiode is affixed near the flow cell, parallel to the plane of the laser. All of the light that is scattered orthogonally by the cell or particle toward one side of the flow cell intersects the RAS photodiode. This gives orthogonal acceptance angles of ⁇ 50°-130°, or a numerical aperture of (NA) of 0.9.
- This high numerical aperture RAS photodiode takes up an entire side face of the flow cell, so no other measurements can be made on this side of the flow cell. However, the face opposite the RAS detector is still available, and can be used to obtain other measurements.
- this side of the flow cell may be used to generate standard fluorescent flow cytometer data, by employing detectors capable of detecting emitted fluorescent light.
- This side may also contain one or more of the following: a collection lens (low numerical aperture) that will image the flow stream in the center of the flow cell; beam splitters and interference filters, to separate several axes of fluorescent information; imaging lenses to focus the light to a detector; and photo multiplier tubes to detect low levels of florescent emissions.
- a lensless light detection system eliminates the need for associated optical elements, such as lenses, stops, and mirrors.
- the resulting light detection system is significantly smaller, lighter, less expensive, and more reliable than conventional light detection systems which rely on such optical elements to direct light beams within the instrument.
- a disadvantage of using a lensless light collection system is that stray light easily gets into the detectors.
- Lenses are conventionally used to direct the light that images the core stream, so that only light that emanates from the core stream gets to a detector.
- Lenses are also use to focus light collected from full cone angles down to a point where a small photodetector is placed.
- the use of a relatively small photodetector minimizes dark current noise, which is proportional to the area of the photodiode.
- dark current noise baseline is not achievable.
- the constant background of light on the photodiodes generates a constant photo-current, in the signal processing electronics. This constant photo-current electronically is analogous to direct current, or DC, thus the lensless system generates a DC light level on each photodiode.
- the preamp circuits In order to keep the outputs at a baseline value when no signal is present there is a local servo loop for each of the preamp circuits associated with the optical channels (EXT, FSL, FSH, RAS). Photodiode dark currents, input bias currents, or offset voltages can cause offsets or drift in the outputs of the preamp circuits.
- the feedback resistor produces a current proportional to the voltage at the output of the second stage.
- the second method of feedback uses an op-amp configured as an integrator to return a proportion of the signal back to the first stage op-amp to reduce the offset levels through the first and second stage op-amps.
- the local servo loops reduce low frequency signals. Equation (1) may be used to determine the corner frequency at which the servo loop will provide (-3dB) attenuation.
- R t Current to Voltage conversion resistor
- R f Servo input resistor
- Hemoglobin concentration is a very common hematology parameter that is generally measured by light absorption at 540 nm.
- red blood cells are generally lysed in a solution containing 1 part whole blood to 250 part solution.
- This solution generally also contains a low level of a cyanide salt (i.e., KCN).
- KCN a cyanide salt
- the cyanide reduces all of the hemoglobin (oxy- and deoxy-) to hemoglobincyanide, which absorbs maximally at 540 nm.
- This absorption measurement is conventionally made in a 1.0 cm square cuvette (NCCLS), but other variants from the standard also work with high correlation to the reference method.
- the hemoglobin concentration is generally measured in an otherwise clear solution, and is always referenced to a clear fluid. Lysis of red cells allow the hemoglobin to be measured in the same fluidic channel as the white blood cells. Alternatively, on some systems, the hemoglobin content is measured in a separate channel.
- hemoglobin (HGB) content is measured in an unconventional manner.
- the method of the present invention involves two separate measurements, which not only combine to give an accurate HGB value, but also verify the accuracy of the dilution that the system has made.
- the first HGB measurement occurs after one part whole blood is diluted 100-fold with a reticulocyte staining solution. This is referred to as the "red cell solution”.
- This reticulocyte staining solution has a specific absorption spectra associated with it.
- the red cell solution is aspirated into a manifold block.
- This block contains at least three light emitting sources, configured to illuminate a cylindrical bore, where the red cell solution is held. Photodetectors are place in line with the light source and the cylindrical bore, with provisions for interference filters. This configuration allows for the measurement at least three different wavelengths of absorption. Three different wavelengths of absorption are used because of the presence of a dye in the reticulocyte staining solution.
- the initial absorption measurements of the red cell solution do not provide as accurate a measurement of HGB concentration as other conventional techniques.
- HCT Hematocrit
- the lytic agent destroys the red blood cell membranes (lysis), releasing HGB to be free in solution. This yields approximately 1 part whole blood to between 15 and 50 parts solution, which is referred to as the "white cell dilution".
- the white cell solution contains all of the dye from the previously measured red cell solution.
- the original concentration of the dye solution has now been diluted out by a factor between 1 : 1 and 1 :4, with an optically clear fluid.
- the white cell solution is aspirated into the manifold block where multiple absorption measurements are made, and a HGB value calculated. From this second HGB value, the HCT, MCHC and MCH can be recalculated. In addition to the HGB value, dye concentration can be measured, and compared to values obtained from the red cell solution. If, within a defined tolerance, the two independent HGB (HCT, MCHC, MCH) measurements match, and the dye ratios between the two solutions match, the instrument has, by measurement, verified all of its dilutions were performed accurately.
- the white blood cell HGB calculations due to the lysis of the RBCs, yields an accurate measurement of the HGB concentration.
- the red blood cell HGB calculations are not as accurate as the white blood cell HGB calculations, because the intact red cell membranes scatter light.
- a comparison of the differences in the absorbances that are due to the reticulocyte staining dye in each measurement permit a check of the accuracy of the dilutions. That is, the sample containing unlysed RBCs (the red cell solution) has a certain level of light absorption which is based on the concentration of the dye in the consumable tube. Later in the cycle, the sample containing lysed RBCs (the white cell solution) is measured for light absorption due to the dye.
- the accuracy of the dilutions may be determined without the need for a separate reference solution.
- the acceptable range of ratios of the HGB measurements is known, either from being printed on a barcode label on the consumable tube or from a code on the consumable tube barcode label that references a value in a table of ratios stored in the PC.
- the HGB measurements should also match within a certain tolerance. Otherwise, a dilution error should be suspected. The only case in which the dilution error would be undetected is the highly unlikely situation in which identical dilution errors are made on both samples .
- the bar code on the consumable tube contains reference information on the expected values of dye absorption for undiluted samples (CAL).
- CAL dye absorption for undiluted samples
- the instrument verifies this information for each lot number in use.
- the red cell solution should have a dye absorption value and an HGB concentration value that fall within a first set of expected ranges. These ranges should be the product of CAL x a first dilution factor (Dl).
- Dl first dilution factor
- the white cell solution should have a dye absorption value and an HGB concentration that fall within a second set of expected ranges.
- D2 second dilution factor
- the instrument of the present invention employs at least two primary method reagents and a third system reagent. These three reagents work together to first count and classify platelets and red cells, and then, after further reagent manipulation, count and classify white blood cells. The counting and classification take place in two discrete phases, and in each of the two discrete phases the blood-reagent mixtures are passed through a flow cytometer and cells in the samples are identified and counted.
- the consumable contains the following:
- New methylene blue in a concentration range from 0.1 to 0.5 grams per liter. This component serves to stain the residual RNA in reticulocytes a blue color. The preferred concentration is 0.3 grams per liter.
- Purafac-A-39-Pric in a concentration range from 0.1 to 0.6 grams per liter. This component acts to modify the normal biconcave shape of red blood cells, by interacting with the cell membrane and the internal hemoglobin, to form spherical cells.
- the preferred concentration is 0.3 grams per liter.
- the buffers and preservatives consist of sodium bicarbonate (preferably 8.0 grams per liter, but can range from 6.0 to 10.0 grams per liter); sodium cloride (preferably at 3.1 grams per liter); Tricine (preferably at 1.8 grams per liter, but ranging from 1.0 to 5.0 grams per liter); di-sodium EDTA (preferably at 1.0 grams per liter, but ranging from 0.5 to 3.0 grams per liter); ethyl paraben (0.3 grams per liter), and methyl paraben (0.2 grams per liter).
- the pH of this solution should be in a slightly basic range between 7.2 to 8.9, with an osmolarity of between 275 and 294 milliosmoles, and a conductivity of between a bicarbonate (preferably 8.0 grams per liter, but can range from 6.0 to 10.0 grams per liter); sodium cloride (preferably at 3.1 grams per liter); Tricine (preferably at 1.8 grams per liter, but ranging from 1.0 to 5.0
- the internal reference particles are to be used at known concentration, and are to act as a cellular surrogate.
- This surrogate must have properties such that the instrument in at least one measurement technique can uniquely distinguish it from the cellular constituents of the sample.
- This cellular surrogate acts as an intra-sample, internal standard for quality control purposes. This assures that the instrument's measurements are performing properly, and that all dilutions have been made properly.
- the preferred embodiment of this surrogate is polystyrene particles. However, other particles, such as fixed cells, pollen, glass, or large colloids could be used, so long as the above conditions are fulfilled.
- the particle size range is preferably from about 1 micron to about 10 microns, and have optical characteristics that uniquely separate them from platelets, white cells and red cells on at least one measurement axis of the instrument.
- Polystyrene particles are commercially available through several manufacturers, such as Seradyn and Duke.
- the preferred size is 4.0 microns, which is easily distinguished from the cellular components of blood along several axes of data collection in the instrument. Choosing this size for the particles allow the particle's concentration to be easily determined.
- the reagent systems within the consumable are sensitive to a percent solids measurement (volume of particle time concentration of particles); thus, the lower the volume chosen, the higher the concentration of particles, and the broader that range of particles concentrations that may be practiced.
- the preferred embodiment is to have the concentration of particles in the consumable at 10,000 per ⁇ l. At this level, the number of polystyrene events counted will be within the same range as the number of white blood cells counted.
- the second method reagent is designated the "lyse".
- the lyse acts to destroy red blood cells while leaving the white blood cells intact. In this fashion, the white blood cells can be analyzed, without interference from the vast numbers of red blood cells.
- This reagent may be packaged in a standard, capped, test tube, preferably in a form that allows it to be used with up to fifty separate consumable tests.
- the lyse preferably contains:
- Saponin which can be used at concentrations between 6.0 and 20.0 grams per liter. The preferred concentration is 18 grams per liter.
- Buffers and preservatives consist of sodium sulfate (preferably at 12.0 grams per liter, but effective in a range of 10.0 - 16.0 grams per liter); disodium EDTA (preferably at 1.0 grams per liter, but effective from 0.5 to 3.0 grams per liter); pro-clin 300 (preferably at a concentration of 0.5 ml per liter); and germabox II (preferably at 1.0 ml per liter).
- This pH of this reagent should be in a range between 4.2 and 5.2, with an osmolarity of between 235 and 285 milliosmoles, and a conductivity of between 14 and 16.
- the system reagent primarily acts as a whole blood diluent, as a sheath solution and as a wash reagent.
- the system reagent is also referred to as "sheath solution”.
- the sheath solution performs several functions, such as whole blood dilution reagent, sheath reagent for flow cytometric analysis, washing reagent for the instrument's hydraulic path, and lytic aide for the lyse reagent to clear red blood cells.
- the sheath is preferably packaged in a container that will hold enough to run fifty separate consumable tests.
- the sheath contains the following: • Phosphate-buffered saline solution (preferably having an osmolarity of 25 milliosmoles, but can be used in a range from 5 to 50 milliosmoles). • Surfactant to aid in both cleaning of internal system components, and to keep internal wetted surfaces from having air bubbles adhere to them.
- the preferred surfactant is Plurafac-A-39, which can be used in a range of 0.1 grams per liter to 0.3 grams per liter, and preferably at a concentration of about 0.1 grams per liter.
- non-ionic surfactants such as could be used.
- a hydraulic system and various mechanisms is used.
- the user presents to the instrument a barcode-labeled consumable tube, as well a properly anti-coagulated whole blood sample.
- the instrument reads the necessary information from the barcode, then automatically aliquots a small amount of whole blood into the consumable, along with a volume of sheath solution.
- This ideally creates a total dilution of one part blood to 100 parts solution (RBC / retic reagent and sheath), but the dilution can range from 1 :50 to 1:5,000.
- a known volume of this solution is then pulled into the instrument from the consumable tube and moved to a position near the entrance to the optical flow cell.
- the solution is then passed, at a slow mass flow rate of less then 0.25 microhters per second by hydrodynamic focusing, through an optical detection system, where light scatter and light absorption for individual red blood cells, platelets and microparticles, as well as light absorption by a dye in the solution, are measured.
- the instrument aliquots a second, larger amount of whole blood into the consumable tube, along with an aliquot of lyse and appropriate amounts of sheath solution, to form a dilution of preferably one part blood to twenty parts solution.
- This dilution can range from 1 : 10 to 1 : 100.
- the intent is to provide a solution that can lyse the red blood cells, but keep intact the white cells of the whole blood sample for a period of at least one minute.
- a known volume of this second solution is then pulled into the instrument from the consumable tube and moved to a position near the entrance to the optical flow cell.
- the solution is then passed, at a moderate mass flow rate of up to about one microliter a second using hydrodynamic focusing, through an optical detection system, where light scatter and light absorption for individual white blood cells, microparticles, and dye are measured.
- the user opens a door of the system, when it is in the READY state, which exposes two open tube slots (figure 7 slot A and figure 7 slot B). In these empty slots, the user places a consumable tube (60), and a whole blood sample, which contains an anticoagulant (i.e. EDTA, Citrate, and Heparin). The user then closes the door.
- the system verifies the presence and identity of the two added tubes, and moves the vial holding carriage (70) so that the consumable is positioned under the mixer (80). Motor 82 position the mixer so that it is in contact with the top of the consumable tube (60). Spinning it in one direction, and then reversing the direction mix the tube. During the spinning process, the bar code label is read which contains lot calibration information.
- the carriage 70 positions the whole blood sample under the mixer, and in a similar manner mixes the whole blood tube so as to ensure sample homogeneity.
- the carriage then potions the whole blood tube underneath the hydraulic needle 11 and pneumatic needle 12.
- the needles By moving motor 81, the needles then pierce the whole blood tube's septum, and continue to penetrate the tube until the both needles come in contact with the surface of the blood. This ensures that the orifice of hydraulic needle 11 is completely immersed, but the orifice of pneumatic vent needle 12 is not. Opening valve 3 , and moving syringe 6 pulls blood pulled into the tip of needle 11.
- Valve 3 is then closed.
- the needles are then retracted from the whole blood patient sample.
- the needles are wiped almost completely clean by the septum.
- the carriage 70 now positions the consumable tube 60 under the needles.
- the needlfes 11 and 12 pierce the septum of consumable tube 60, and penetrate the tube until the both needles come in contact with the surface of the reagent.
- Valves 5 and 3 are then opened, while syringe 7, which is filled with the system diluent begins to dispense. This pushes the blood from the tip of the needle 11 and a volume of sheath fluid into the consumable, which is now represented by 61. While dispensing, motor 81 has moved the needles 11 and 12 up so as to prevent the orifice of needle 12 from coming into contact with solution. This solution is now fairly homogeneous, but to ensure homogeneity, valves 3 and 5 are closed, and the needles are withdrawn from 61, which is then moved to the mix position under 80, and mixed by spinning, as described above. During this process valve 3 and valve 5 open, and syringe 7 creates an air gap at the tip of the needle.
- Consumable tube 61 is next place under needles 11 and needle 12.
- the needles llandl2 pierce the septum of the consumable, and penetrate the tube until the both needles come in contact with the surface of the solution.
- Syringe 7 then aspirates a know volume of solution into the tip of the needle.
- the needles 11 and 12 are then withdrawn from the solution, but are not removed from consumable tube 61, by motor 81.
- Syringe 7 the pulls this slug through the HGB module 10, where it is evaluated for absorption at four separate wavelength of light.
- Such spectral data is represented in figure 22.
- the volume of the slug can also be evaluated here by evaluating the absorption values of the detector channels, which change as the air gaps on each side of the slug pass through.
- Syringe 7 continues to pull the slug past valve 3, and positions it close to valve 5.
- Valve 3 closes while valve 2 opens, at which point syringe 7 reverses it's direction and pushes the slug through valve 2, to the tip of the flow cell assembly 20.
- Valve 2 then closes, and valve 4 opens, which allows syringes 6 and 7 to be filled form the system diluent reservoir 8.
- Valve 5 then closes, valve 1 opens, and syringes 6 and 7 dispense very slowly.
- the sheath solution of syringe 7 is dispensed through 21, and the slug of diluted whole blood is pushed by syringe 6 through 20.
- This stream of fluid passes completely through flow cell 22, and into the tubing of 23, which is affixed to the flow cell 22 by flow cell cap 24. This leads path leads to the system waste 9.
- optical assembly 41 illuminates the cells of the diluted whole blood individually.
- the cells pass through an optical focal point 34 of the laser 30, which maximize the incident power upon the cells. This narrow focus is generated by lens 33.
- Light effected by the cell passages through focal point 34 changes the quantities of light that is incident upon photodiodes 42, 43, 44 and 45. These changes are captured by the signal processing electronics of figure 26, and stored via the system electronics of figure 25. Examples of data collected in this manor are shown FIG 10 A - 10 D.
- Fig 10 A shows data which has been collected for the forward scatter low (FSL) signal, from detector 45.
- 10 B shows data, which has been collected for the extinction or axial light loss (EXT) signal, from detector 44.
- Fig 10 C shows data, which has been collected for the right angle scatter (RAS) signal, or detector 42.
- Fig 10 D shows pulse width data which has been collected from the forward scatter low (FSL) signal, or detector 45, but measure the time of flight, or the time the cell is in front of the laser beam.
- the peak's shown by A represent platelet events
- the peak's shown by B represents latex particle events
- the peak's shown by C represents red blood cell events.
- RNA staining dye such as new methylene blue
- the data can be further analyzed by looking only at the red blood portions, C, in Figures 10 A - 10 D to evaluate reticulocytes. Histogram of such analysis are shown in figures 14 A and 14 B, and a scatter plot is shown in Figure 15.
- Figure 15 shows a calculated 1.72-% population, which are reticulocytes. This compares favorably with a manual reference method of 1.60-% reticulocyte count.
- the needles 11 and 12 are withdrawn from the consumable 61.
- Carriage 70 moves the whole blood sample so it is positioned under the needles 11 and 12.
- Moving motor 81 the needles pierce the whole blood tube's septum, and continue to penetrate the tube until the both needles 11 and 12 come in contact with the surface of the blood. This ensures that the orifice of hydraulic needlel 1 is completely immersed, but the orifice of pneumatic vent needlel2 is not. Opening valve 3, and moving syringe 6, causes blood is pulled into the tip of needle 11. Ideally, 100 microhters will be aspirated for this portion. Valve 3 is then closed. Needles 11 and 12 are then retracted from the whole blood patient sample. As the needles 11 and 12 withdraw through the septum, the needles are wiped almost completely clean by the septum.
- the carriage 70 now positions the consumable tube 61 under the needles 11 and 12.
- needles 11 and 12 pierce the septum of consumable 61 , and penetrate the tube until the both needles 11 and 12 come in contact with the surface of the solution.
- Valve 5 and valve 3 are opened, while syringe 7, which is filled with the system diluent begins to dispense. This pushes the 100 microhters of blood through the tip of the needle 11.
- a volume of sheath fluid follows the whole blood into the consumable, which is now represented by 62.
- dispensing motor 81 has moved the needles up, so as to prevent the orifice of needle 12 from coming into contact with solution.
- Needles 11 and 12 are now extracted from consumable tube 62, and carriage 70 moves the positions of figure 7 slot C, which contains a tube of lysing reagent, under needles 11 and 12.
- Moving motor 81 the needles 11 and 12 pierce the lyse tube's septum, and continue to penetrate the tube until the both needles 11 and 12 come in contact with the surface of the lyse. Opening valve 3, and moving syringe 6, pulls lyse into the tip of needle 11. Ideally, 100 microhters of lyse will be aspirated for this portion, which is then followed by an air gap. Removing the needle 11 from the solution, then moving syringe 6 creates this air gap. Valve 3 is then closed. Needles 11 and 12 are then retracted from the lyse tube. As the needles withdraw through the septum, the needles are wiped almost completely clean by the septum.
- Carriage 70 positions the consumable 62 under the needles 11 and 12. Valve 5 and 3 are then open, while syringe 7, which is filled with the system diluent begins to dispense. This pushes the 100 microhters of lyse through the tip of the needle 11. A volume of sheath fluid follows this into the consumable, which is now represented by 63. While dispensing, motor 81 has moved the needles up, so as to prevent the orifice of needle 12 from coming into contact with the solution. The solution in consumable tube 63 fairly homogeneous, but to ensure homogeneity, valves 3 and 5 are closed, and the needles are withdrawn from 63, which is then moved to the mix position under 80, and mixed by spuming, as describe above. During these process valves 3 and 5 open, and syringe 7 creates an air gap at the tip of the needle. Consumable 63, is place under the needles 11 and 12. Moving motor 81, needles
- Absorption data can be shown by figure 19 A if cyanide was used in the lysing agent, figure 19 B if only a lytic agent was used, and by figure 21, if the cell lysis occurred in the presense of a reticulocyte stain.
- the volume of the slug can also be evaluated here by evaluating the absorption values of the detector channels, which change as the air gaps on each side of the slug pass through.
- Syringe 7 continues to pull the slug past valve 3, and close to valve 5.
- Valve 3 closes, and valve 2 opens, at which point syringe 7 reverses it's direction and pushes the slug through valve 2, to the tip of the flow cell assembly 20.
- Valve 2 then closes, and valve 4 opens, which allows syringes 6 and 7 to be filled form " the system diluent reservoir 8.
- Valve 5 then closes, valve 1 opens, and syringes 6 and 7 dispense very slowly.
- the sheath solution of syringe 7 is dispensed through 21, and the slug of lysed whole blood is pushed by syringe 6 through 20.
- This stream of fluid passes completely through flow cell 22, and into the tubing of 23, which is affixed to the flow cell 22 by flow cell cap 24. This leads path leads to the system waste 9.
- optical assembly 41 illuminates the cells of the diluted whole blood individually.
- the cells pass through an optical focal point 34 of the laser 30, which maximize the incident power upon the cells. This narrow focus is generated by lens 33.
- Light effected by the cell passages through focal point 34 changes the quantities of light that is incident upon photodiodes 42, 43, 44 and 45. These changes are captured by the signal processing electronics of figure 26, and stored via the system electronics of figure 25. Examples of data collected in this manor are shown in figures 12 A - 12 F.
- Fig 12 A shows data which has been collected for the extinction or axial light loss
- EXT right angle scatter
- the peak shown for A represent lymphocyte events
- the peak shown for B represents monocyte events
- the peak shown for C represents granulocyte events
- the peak shown for D represent latex events.
- This same data is shown in a scatter plot form in figures 13 A and 13 B.
- the dots in circle D represent the latex particle events
- circle D also represent the latex particle events. Scatter plot data could be represented by any pair- of the up to four detectors, and time of flight.
- Figure 16 A shows the histogram of data where A represents lymphocytes, B monocytes, C neutrophils, and D eosinophils.
- Figure 17 shows the same data as Figure 16, but in scatter plot form. The encircle populations of A represents lymphocytes, B monocytes, C neutrophils, and D eosinophils. Any pair of up to four detectors and time of flight could represent by scatter plot data, but to distinguish eosinophils, right angle scatter is necessary.
- syringe 7 is charged with fluid from reagent reservoir 8, by the opening of valve 4.
- Valve 4 closes, 3 and 5 open, which allows syringe 7 to clean the samples lines by back-flushing fluid into the consumable tube 63 through needle 11.
- Needle 12 acts to vent the tube through filter 13, to atmosphere. This yields a consumable tube that is of the configuration shown in 64.
- Carriage 70 now moves to it's home position, where the user can open the system door, and remove the tubes from figure 7 slots A and B. The consumable is now a waste tube, and is discarded.
Abstract
Description
Claims
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
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AU6340501A AU6340501A (en) | 2000-06-02 | 2001-05-24 | Flow cytometry-based hematology system |
EP01937695A EP1297334B1 (en) | 2000-06-02 | 2001-05-24 | Method of using a flow cytometry-based hematology system |
BRPI0111373A BRPI0111373B8 (en) | 2000-06-02 | 2001-05-24 | multi-purpose blood sample reaction vessel for use in a flow cytometry-based hematology system, and flow cytometry system |
CA2410894A CA2410894C (en) | 2000-06-02 | 2001-05-24 | Flow cytometry-based hematology system |
MXPA02011826A MXPA02011826A (en) | 2000-06-02 | 2001-05-24 | Flow cytometry-based hematology system. |
AT01937695T ATE546722T1 (en) | 2000-06-02 | 2001-05-24 | METHOD OF USING A FLOW CYTOMETRY BASED HAEMATOLOGICAL SYSTEM |
JP2002502436A JP4078600B2 (en) | 2000-06-02 | 2001-05-24 | Hematology device based on flow cytometry |
ES01937695T ES2378352T3 (en) | 2000-06-02 | 2001-05-24 | Procedure to analyze a blood sample or a sample of a blood derivative using a hematological system based on flow cytometry |
Applications Claiming Priority (4)
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US20884900P | 2000-06-02 | 2000-06-02 | |
US60/208,849 | 2000-06-02 | ||
US09/715,593 US6784981B1 (en) | 2000-06-02 | 2000-11-17 | Flow cytometry-based hematology system |
US09/715,593 | 2000-11-17 |
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WO2001094938A1 true WO2001094938A1 (en) | 2001-12-13 |
WO2001094938A9 WO2001094938A9 (en) | 2002-10-10 |
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PCT/US2001/016814 WO2001094938A1 (en) | 2000-06-02 | 2001-05-24 | Flow cytometry-based hematology system |
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US (4) | US6784981B1 (en) |
EP (1) | EP1297334B1 (en) |
JP (1) | JP4078600B2 (en) |
AT (1) | ATE546722T1 (en) |
AU (1) | AU6340501A (en) |
BR (1) | BRPI0111373B8 (en) |
CA (1) | CA2410894C (en) |
ES (1) | ES2378352T3 (en) |
MX (1) | MXPA02011826A (en) |
WO (1) | WO2001094938A1 (en) |
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EP1297334A4 (en) | 2006-05-17 |
WO2001094938A9 (en) | 2002-10-10 |
USRE42143E1 (en) | 2011-02-15 |
MXPA02011826A (en) | 2004-05-17 |
BRPI0111373B1 (en) | 2015-09-22 |
BR0111373A (en) | 2004-02-10 |
ES2378352T3 (en) | 2012-04-11 |
US6784981B1 (en) | 2004-08-31 |
EP1297334A1 (en) | 2003-04-02 |
AU6340501A (en) | 2001-12-17 |
US20030030783A1 (en) | 2003-02-13 |
EP1297334B1 (en) | 2012-02-22 |
US7324194B2 (en) | 2008-01-29 |
ATE546722T1 (en) | 2012-03-15 |
JP2004506876A (en) | 2004-03-04 |
CA2410894C (en) | 2013-04-16 |
BRPI0111373B8 (en) | 2021-07-27 |
JP4078600B2 (en) | 2008-04-23 |
CA2410894A1 (en) | 2001-12-13 |
US20060203226A1 (en) | 2006-09-14 |
US7064823B2 (en) | 2006-06-20 |
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