Flow cytometry-based hematology system

The present invention is a flow cytometry-based hematology system useful in the analysis of biological samples, particularly whole blood or blood-derived samples. The system is capable of determining at least a complete blood count (CBC), a five-part white blood cell differential, and a reticulocyte count from a whole blood sample. The system preferably uses a laser diode that emits a thin beam to illuminate cells in a flow cell and a lensless optical detection system to measure one or more of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight measurements produced by the cells. The lensless optical detection system contains no optical components, other than photoreactive elements, and does not include any moving parts. Finally, 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 reference particles, which act as internal standards to ensure that the dilutions made during processing of the samples have been carried out correctly, and to ensure that the instrument is working properly. The present invention also relates to methods for using the system.

 

What is claimed is:

1. A flow cytometry-based hematology system for classifying and counting biological cells in a blood sample or blood-derived sample, comprising:

a hydraulic system including a flow cell, said hydraulic system being capable of mixing said blood sample or blood-derived sample with the contents of one or more of a disposable vessel, a lyse tube or a sheath tube, and being capable of moving the contents of one or more of said disposable vessel, said lyse tube, said sheath tube, or a mixture of said blood sample or said blood-derived sample with one or more of said contents of said disposable vessel, said lyse tube and said sheath tube through said flow cell;

a diode laser, said diode laser emitting light to irradiate cells present in said flow cell, said cells being derived from said blood sample or said blood-derived sample;

a lensless light detection system, said lensless light detection system having a first portion capable of detecting one or more of axial light loss, low-angle scattered light and high-angle scattered light, and a second portion capable of detecting right angle scattered light scattered at a high numerical aperture, said second portion being physically distinct from said first portion, said first portion and said second portion being capable of converting said axial light loss, said low-angle scattered light, said high-angle scattered light, and said right angle scattered light into electrical signals;

a signal processor, said signal processor being capable of analyzing said electrical signals generated from said first portion and said second portion of said lensless light detection system, and generating data therefrom; and

a servo circuit capable of correcting for the influence of dark current noise and non-scattered light incident on said lensless light detection system.

2. A flow cytometry-based hematology system for classifying and counting biological cells in a blood sample or blood-derived sample, comprising:

a hydraulic system including a flow cell, said hydraulic system being capable of mixing said blood sample or blood-derived sample with the contents of one or more of a disposable vessel, a lyse tube or a sheath tube, and being capable of moving the contents of one or more of said disposable vessel, said lyse tube, said sheath tube, or a mixture of said blood sample or said blood-derived sample with one or more of said contents of said disposable vessel, said lyse tube and said sheath tube through said flow cell;

a diode laser, said diode laser emitting light to irradiate cells present in said flow cell, said cells being derived from said blood sample or said blood-derived sample;

a lensless light detection system, said lensless light detection system having a first portion capable of detecting one or more of axial light loss, low-angle scattered light and high-angle scattered light, and a second portion capable of detecting right angle scattered light scattered at a high numerical aperture, said second portion being physically distinct from said first portion, said first portion and said second portion being capable of converting said axial light loss, said low-angle scattered light, said high-angle scattered light, and said right angle scattered light into electrical signals; and

a signal processor, said signal processor being capable of analyzing said electrical signals generated from said first portion and said second portion of said lensless light detection system, and generating data therefrom;

a reservoir for a sheath solution;

at least one syringe connected to said reservoir, said at least one syringe being driven by a motor;

a single HGB module, said HGB module being connected on an inlet side to a sample inlet;

said sample inlet being connected to said flow cell by a single flow path passing through said HGB module;

a single flow cell, said single flow cell being connected to said HGB module and said at least one syringe on an inlet side of said flow cell, and to a waste container on an outlet side of said flow cell.

3. The flow cytometry system of claim 2, wherein said first portion comprises a photodetector array arranged on a plurality of electrically distinct integrated circuit chips.

4. The flow cytometry system of claim 3, wherein said photodetector array has at least three separate and electrically distinct light detection elements.

5. The flow cytometry system of claim 4, wherein said elements are made from a single piece of photoreactive material.

6. The flow cytometry system of claim 4, further comprising a Mie mask placed over said photodetector array.

7. The flow cytometry system of claim 4, wherein:

a first one of said light detection elements is capable of detecting axial light loss;

a second one of said light detection elements is capable of detecting forward scattered light scattered at an angle between about 1 and about 3 degrees; and

a third one of said light detection elements is capable of detecting forward scattered light scattered at an angle between about 4 and about 9 degrees.

8. The flow cytometry system of claim 4, wherein:

said first, second and third light detection elements are aligned on a first axis that is substantially orthogonal to a second axis defined by a line of travel of cells through a flow cell of said flow cytometer and further is substantially orthogonal to a third axis defined by a line of travel of laser light incident on said cells in said flow cell; and

said second axis and said third axis are substantially orthogonal to each other.

9. The flow cytometry system of claim 2, wherein said second portion comprises a high numerical aperture light detector capable of collecting light scattered at an angle between about 50 and about 130 degrees.

10. The flow cytometry system of claim 7, further comprising a servo circuit capable of correcting for the influence of dark current noise and non-scattered light incident on said lensless light detection system.

11. The flow cytometry system of claim 7, wherein said diode laser is capable of emitting a laser light beam having a height between about 0.1 and about 10 .mu.m, and a width between about 0.1 and about 200 .mu.m.

12. The flow cytometry system of claim 7, further comprising a fluorescent light detector capable of detecting light emitted from a fluorescent compound present in said flow cell upon illumination of said fluorescent compound by said diode laser.

13. The flow cytometry system of claim 7, further comprising a bar code reader, said bar code reader being capable of reading information encoded on a bar code label present on a surface of said disposable vessel;

said information including one or more of type of test, lot number of reference particles present in said disposable vessel, expiration date of said disposable vessel, a serial number of said disposable vessel, a reference particle concentration, a mean value of axial light loss and coefficient of variation of axial light loss for said reference particles, a mean value of low-angle forward light scatter and coefficient of variation of low-angle forward light scatter for said reference particles, a mean value of high-angle forward light scatter and coefficient of variation of high-angle forward light scatter for said reference particles, a mean value of right angle light scatter and coefficient of variation of right angle light scatter for said reference particles, a mean value of time-of-flight and coefficient of variation of time-of-flight for said reference particles, absorbance of a sample mixture, transmission of a sample mixture, reflectance of a sample mixture, or fluorescence of a sample mixture.

14. The flow cytometry system of claim 2, wherein said hydraulic system comprises not more than one flow cell.

15. The flow cytometry system of claim 2, wherein said hydraulic system further comprises a plurality of valves to control flow through said hydraulic system, and wherein the number of said valves does not exceed five.

16. A lensless light detection system for a flow cytometer, said lensless light detection system comprising one or more detectors fixed in space relative to said flow cytometer and capable of collecting discrete light signals produced by cells passing through a laser beam in said flow cytometer in the absence of optical elements, and further comprising a servo circuit capable of correcting for the influence of dark current noise and non-scattered light incident on said lensless light detection system.

17. A method for analyzing a blood sample or a blood-derived sample using a flow cytometry-based hematology system, comprising the steps of:

a) mixing a first portion of said blood sample or blood-derived sample with a reagent in a disposable vessel containing a known number of reference particles to form a red cell solution, said reference particles having one of a plurality of predetermined diameters such that when said reference particles are illuminated by light, said light is scattered such that said scattered light falls into one of a plurality of predetermined light scatter channels;

b) standardizing said red cell solution and said flow cytometry-based hematology system using said reference particles by measuring at least one of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight for each of said reference particles, and comparing said measurements to predetermined values obtained for said reference particles;

c) counting at least a portion of red blood cells, reticulocytes, and platelets present in said red cell solution;

d) measuring at least one parameter from the group measured in step b) for each of one or more cells in said red cell solution, to produce a red cell solution measurement value for each parameter for one or more of said red blood cells, reticulocytes, and platelets from said sample;

e) mixing a second portion of said blood sample or blood-derived sample with a lyse solution in said disposable vessel containing a known number of reference particles to form a white cell solution;

f) counting at least a portion of white blood cells present in said white cell solution;

g) measuring at least one parameter from the group measured in step b) for each of one or more cells in said white cell solution, to produce a white cell solution measurement value for each parameter for said white blood cells; and

h) analyzing said red cell solution measurement values and said white cell solution measurement values to classify each cell.

18. The method of claim 17, wherein said low-angle forward scattered light is light scattered at an angle between about 1 and about 3 degrees.

19. The method of claim 17, wherein said high-angle forward scattered light is light scattered at an angle between about 4 and about 9 degrees.

20. The method of claim 17, where said right angle scattered light is scattered at an angle between about 50 and about 130 degrees.

21. The method of claim 17, wherein said low-angle scattered light is light scattered at an angle between about 1 and about 3 degrees, said high-angle scattered light is light scattered at an angle between about 4 and about 9 degrees, and said right angle scattered light is scattered at an angle between about 50 and about 130 degrees.

22. The method of claim 17, further comprising measuring a hemoglobin content of said blood sample or blood-derived sample.

23. The method of claim 17, further comprising measuring an amount of light emitted from a fluorescent compound present in said blood sample or blood-derived sample after said reagent is mixed with said blood sample or blood-derived sample.

FIELD OF THE INVENTION

This invention relates in general to bioparticle analysis, and more specifically to flow-cytometry-based methods and devices for performing automated blood cell analysis.

BACKGROUND OF THE INVENTION

Mammalian peripheral blood usually contains three major classifications of blood cells--red blood cells ("RBCs"), white blood cells ("WBCs"), and platelets ("PLTs"). 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 include blood coagulation, osmolality maintenance, immune surveillance, and a multitude of other functions.

Mammals usually have anywhere from 2-10.times.10.sup.12 RBCs per liter. RBCs are responsible for oxygen and carbon dioxide transport within the circulatory system. In many mammals, including humans, 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. The RBCs contain high concentrations of hemoglobin, a heme-containing protein which performs the dual roles of oxygen and carbon dioxide transport. Hemoglobin is responsible for the overall red color of blood, due to the presence of iron in the heme molecule. In the present application, the terms "erythrocytes", "red blood cells", "red cells", and "RBCs" are used interchangeably to refer to the hemoglobin-containing blood cells present in the circulation as described above.

In addition to mature RBCs, immature forms of red blood cells can often be found in peripheral blood samples. A slightly immature RBC is referred to as a reticulocyte, and the very immature forms of RBCs are broadly classified as nucleated red blood cells (NRBCs). Higher level non-mammalian animals, such as birds, reptiles, and amphibians, have exclusively nucleated RBCs in their blood.

Reticulocytes are red blood cell precursors that have completed most of the normal red cell development stages in bone marrow, and have expelled their nuclei. The last portion remaining to leave the reticulocyte before it becomes a truly mature RBC is transfer RNA. 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 may be 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 anemic patients provides evidence that their bone marrow is functioning, and attempting to make up for the red blood cell deficit. If few or no reticulocytes are detectable in these patients, the bone marrow is not adequately responding to the red blood cell deficit.

White blood cells (also called "leukocytes") are the blood-borne immune system cells that destroy foreign agents, such as bacteria, viruses, and other pathogens that cause infection. WBCs exist in peripheral blood in very low concentrations as compared to red blood cells. Normal concentrations of these cells range from 5-15.times.10.sup.9 per liter, which about is three orders of magnitude less than red blood cells. These cells are generally larger than RBCs, having diameters between 6 to 13 microns, depending on the type of white cell and the species. Unlike RBCs, there are a variety of white blood cell types that perform different functions within the body. In this application, the terms "white blood cells", "white cells", "leukocytes", and "WBCs" 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 cells in blood is important in the detection and monitoring of a variety of physiological disorders. For example, elevated numbers of abnormal white blood cells may indicate leukemia, which is an uncontrolled proliferation of a myelogenous or a lymphogenous cell. Neutrophilia, or an abnormally high concentration 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 further sub-classified as either lymphocytes or monocytes. Measurements of the percentages in the blood of the two major WBC classifications (granulocytes and mononuclear cells) comprise a two-part WBC differential count (or two-part differential). Measurements of the components of these subclassifications (neutrophils, eosinophils, basophils, lymphocytes, and monocytes), produce a five-part WBC differential count (or five-part differential).

Neutrophils are the most prevalent of the granulocytes and of the five major subclasses 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 at a neutral pH. 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 second 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 are short-lived 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 specifically recognize foreign antigens and in response divide and differentiate to form effector cells. The effector cells may be B lymphocytes or T lymphocytes. B lymphocytes secrete large amounts of antibodies in response to foreign antigens. T lymphocytes exist in two main forms--cytotoxic T cells, which destroy host cells infected by infectious agents, such as viruses, and helper T cells, which stimulate antibody synthesis and macrophage activation by releasing cytokines. Lymphocytes have no granules in their cytoplasm, and their nucleus occupies a large majority of the cell volume. The thin area of cytoplasm outside the nucleus of lymphocytes can be stained with a nucleic acid stain, since it contains RNA. Many lymphocytes differentiate into memory B or T cells, which are relatively long-lived and respond more quickly to foreign antigen than naive B or T cells.

Monocytes are immature forms of macrophages that, in themselves, have little ability to fight infectious agents in the circulating blood. However, when there is an infection in the tissues surrounding a blood vessel, these cells leave the circulating blood and enter the surrounding tissues. The monocytes then undergo a dramatic morphological transformation to form macrophages, increasing their diameter as much as fivefold and developing large numbers of mitochondria and lysosomes in their cytoplasm. The macrophages then attack the invading foreign objects by phagocytosis and activation of other immune system cells, such as T cells. Increased numbers of macrophages are a signal that inflammation is occurring in the body.

Platelets are found in all mammalian species, and are involved in blood clotting. Normal animals will generally have between 1-5.times.10.sup.11 platelets per liter. These cellular particles are usually much smaller than RBCs, having a diameter between 1 and 3 .mu.m. Platelets are formed as buds from the surfaces of megakarocytes, which are very large cells found in the bone marrow. The megakaryocytes do not themselves leave the marrow to enter the blood circulation; rather, buds form on the surface, pinch off and enter the circulation as platelets. Like RBCs, platelets lack nuclei and thus cannot reproduce. Functionally, platelets 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. For example, abnormally low platelet counts may be the cause of a clotting disorder.

Collectively, the counting and sizing of RBCs, the counting of WBCs, and the counting of platelets is referred to as a complete blood count ("CBC"). The separation of white blood cells into the five major classifications (i.e., neutrophils, eosinophils, basophils, lymphocytes, and monocytes) and their quantification on a percent basis is referred to as a five-part differential. The separation of white blood cells into two major classifications, granular and agranular leukocytes, and their quantification on a percent basis is referred to as a two-part differential. The categorizing of red blood cells into two classifications, mature red blood cells and reticulated red blood cells, on a percent basis is referred to as a reticulocyte count.

The determination of a CBC, with a five-part differential and a reticulocyte count, is 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 medical and veterinary clinical laboratories around the world. These three tests have for many years been performed using a microscope, centrifuge, counting chamber, slide, and appropriate reagents. However, the skills necessary to perform these test manually are rare and require years of training. Furthermore, 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 impedance particle counters appeared.

An impedance particle counter counts individual cells or cell clumps, based on a change in impedance across a narrow orifice as a cell passes through. Conventionally, an impedance counter consists of two chambers, each filled with a saline solution and connected via a small orifice. The sample containing cells is introduced into one chamber and passed to the second chamber through the orifice. In the presence of a constant voltage across the orifice, a cell passing individually through the orifice displaces a volume of saline solution and thereby alter the impedance across the orifice. The size of the cell may 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.

As impedance technology evolved, automated cell counters were developed that were able to count RBCs, WBCs, and platelets simultaneously on the same instrument. This automation proved to be a great labor savings for clinical laboratories. However, these systems were unable to distinguish among all the different types of white blood cells, and thus were unable to provide a five-part differential count. Furthermore, these instruments were also unable to distinguish between reticulocytes and normal red blood cells, and thus could also not provide a reticulocyte count.

Flow cytometry is a powerful method of analysis that is able to determine the cellular content of various types of samples, and in particular samples that contain living cells. In clinical applications, flow cytometers are useful for lymphocyte counting and classification, for immunological characterization of leukemias and lymphomas, and for cross-matching tissues for transplants. In most flow cytometry techniques, 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. Different types of cells produce different types of scattered light. The type of scattered light produced may depend on the degree of granularity, the size of the cell, etc. Cells in a fluid solution may also 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. In this fashion, information about the surface components of the cell can be obtained. Examples of such fluorescent molecules include FITC (fluorescein isothiocyanate), TRITC (tetramethyl rhodamine isothiocyanate), Cy3, Texas Red (sulforhodamine 101), and PE (phycoerythrin). In addition, intracellular components of the cell, such as nucleic acids, may be stained by fluorescent compounds, and subsequently detected by fluorescence. Examples of such compounds include ethidium bromide, propidium iodide, YOYO-1, YOYO-3, TOTO-1, TOTO-3, BO-PRO-1, YO-PRO-1, and TO-PRO-1. Cells may also be stained with dyes that label particular cellular components, and the absorbance of the dye bound to the cells measured.

Blood cell measurements made using flow cytometry often require two separate measurements--one to measure the RBCs and platelets, and the other to measure WBCs. The reason for separate measurements is that the RBCs are present in the blood at a much higher concentration than other blood cell types, and thus detection of other cell types in the presence of RBCs requires that the RBCs either be removed or large volumes of sample be measured. Alternatively, these cells may be distinguished on the basis of immunochemical staining of particular cell surface antigens and/or differential cell type staining. Thus, U.S. Pat. No. 5,047,321 (Loken et al.) describes a single step method for the analysis of blood cells that uses at least two fluorescent dyes and a labeled cell marker to differentiate the cell types. However, unless two separate dilutions of the same sample are performed, the method 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.degree.) 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.degree.) is useful in determining cell size. The ability to measure cell size depends on the wavelength employed and the precise range of angles over which light is collected. For example, material within cells having a strong absorption at the illuminating wavelength may interfere with size determination because cells containing this material produce smaller forward scatter signals than would otherwise be expected, leading to an underestimate of cell size. In addition, differences in refractive index between the cells and the surrounding medium may also influence the small-angle scatter measurements.

In addition to forward scattered light, cells having a high degree of granularity, such as granulocytes, scatter incident light at high angles to a much greater degree than cells with low granularity, such as lymphocytes. Different cell types may be distinguished on the basis of the amount of orthogonal light scatter (also referred to herein as right angle side scatter) they produce. As a result, 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.

Additionally, eosinophils may be distinguished from other granulocytes and lymphocytes on the basis of polarization measurements of right angle side scatter. Normally, incident polarized light is scattered orthogonally and remains polarized. However, eosinophils cause incident polarized light scattered orthogonally to become depolarized to a greater degree than other cells. This higher degree of depolarization permits the specific identification of eosinophil populations in blood samples. Copending U.S. patent application Ser. No. 09/507,515, filed Feb. 18, 2000, and incorporated herein in its entirety, discloses an improved high numerical aperture flow cytometer that employs a high angle side scatter light detector to detect eosinophils in a blood sample. However, the use of a high angle side scatter light detector (at angles of about 130.degree.) enables greater discrimination of eosinophils in blood cell populations. In contrast to conventional systems, the system described in copending U.S. patent application Ser. No. 09/507,515 does not require the incident light to be unpolarized.

U.S. Pat. No. 5,492,833 (Rodriguez et al.) discloses a method for distinguishing erythrocytes from reticulocytes by treating the cells with a ghosting reagent, then measuring light scatter. U.S. Pat. No. 5,559,037 (Kim et al.) discloses a method for counting nucleated red blood cells (NRBCs) by first lysing red blood cells, then exposing the NRBCs to a nuclear stain and measuring fluorescence and light scatter. U.S. Pat. No. 5,733,784 (Studholme et al.) teaches a method for measuring the reticulocyte concentration in a blood sample that uses a reticulocyte staining reagent that is incubated for a period between 15 minutes and 4 hours before light scatter measurements are taken. U.S. Pat. No. 5,879,900 (Kim et al.) discloses a method for simultaneous and quantitative analysis of damaged WBCs, NRBCs, and WBC subpopulations that employs multi-dimensional light scatter and fluorescence measurements. U.S. Pat. No. 5,891,734 (Gill et al.) 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.

Hemoglobin (HGB) concentration is a common hematology parameter that is generally measured by light absorption at 540 nm. To accomplish this measurement, red blood cells are generally lysed in a solution containing 1 part whole blood to 250 parts lyse solution. This solution may also contain a low level of a cyanide salt (i.e., KCN). The cyanide reduces the hemoglobin in the blood (oxyhemoglobin and deoxyhemoglobin) to hemoglobincyanide, which absorbs maximally at 540 nm. This absorption measurement is conventionally made in a 1.0 cm square cuvette (NCCLS Standard No. H9-A), but other variants from the standard also work with high correlation to the reference method.

In conventional hematology instruments, the hemoglobin concentration is generally measured in an otherwise clear solution, and is 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 may be measured in a separate channel.

To obtain meaningful information about the numbers and types of cells in a biological sample, or of the concentration of markers on cell surfaces, the samples must be standardized with respect to the amount of light scatter, fluorescence or impedance associated with standardized populations of the cells. In addition, 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 latex beads) or with cells or other biological material (e.g., pollen, fixed cells, or stained nuclei). These standardization materials are desirably extremely uniform in size, and contain precise amounts of fluorescent molecules to serve in calibrating the photomultiplier tubes used in detection of fluorescent probes. However, the calibration procedures are lengthy and complicated, and require extensive training to perform properly. Consequently, these calibration procedures are typically performed only once at the beginning of the analysis. Changes in the instrument or in the sample may alter the performance of the instrument.

U.S. Pat. No. 5,627,037 (Ward et al.) 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 containing 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 number of cells and the number of microparticles provide 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.

U.S. Pat. No. 5,380,663 (Schwartz et al.) 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. U.S. Pat. No. 5,451,525 (Shenkin et al.) 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.

Flow cytometry techniques that took advantage of the light scattering characteristics of cells were applied beginning in the early 1970's to perform white cell differential analysis, in combination with CBC determination. Automated reticulocyte analysis was developed in the 1980's. However, these early systems did not perform a CBC or white blood cell differential. Eventually, manufacturers like Technicon (Bayer), Coulter (Beckman-Coulter) and Abbott incorporated reticulocyte counting with their automated CBC/white cell differential systems, in such high-end hematology systems as the Technicon (Bayer) H*3, Bayer Advia 120.RTM., Coulter STKS.RTM., Coulter GenS.TM., and Abbott CellDyn 3500 and CellDyn 4000. These high-end instrument systems are capable of measuring all of the parameters for a complete hematology analysis that are clinically important for patient assessment, namely, CBC, five-part WBC differential and reticulocyte count.

However, these instruments require complex and expensive optical systems to make such measurements. Furthermore, to reduce the effects of their high initial costs, these hematology instruments are designed to be high-throughput machines. Consequently, multiple different specimen samples are often processed simultaneously, and these samples require different combinations of treatments and analyses. For example, analysis of red blood cells and white blood cells require different reagent systems, and are usually optimized in such instruments by using completely different hydraulic paths for each type of analysis. As a result, to simultaneously perform both red blood cell and white blood cell analyses for a given sample, completely separate and distinct hydraulic paths are created for each type of sample. This combination of multiple sample hydraulic paths creates an undesirably high level of complexity, due to the requirement for large numbers of valves, vacuum lines, reaction chambers, syringes, vacuum pumps, miniature orifices, and the like.

In addition, a significant amount of training is required to learn how to use these instruments properly. Furthermore, these instrument systems require service several times a year, at a minimum. As a result, the high costs associated with purchasing, operating and maintaining these high-end instruments make them financially inaccessible to small clinics and doctor's offices. Doctors can obtain limited access to these instruments by contracting with commercial laboratories that will pick up blood samples from the clinic or doctor's office and transport the samples back to the laboratory for analysis on these high-end hematology systems. The results are then sent to the doctor. This process is very time-consuming, and usually the doctor will not have the results until the following day, at the earliest.

Thus, if a doctor desires a complete hematology assessment (CBC, five-part WBC differential, and reticulocyte count) quickly, the in-office laboratory must perform some combination of manual methods. 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. Thus, to obtain values for these parameters, 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 advanced by incorporating flow cytometric techniques and other improvements, the technology used in the low-end systems has changed very little. These low-end instruments 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 impedance counters 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.

Low-end impedance instruments used in small clinics and doctor's offices generally use bulk reagent packs that are intended for larger laboratories. Since the number of samples run on these instruments is low compared to their laboratory counterparts, significant volumes of reagents are wasted in performing startup and shutdown cycles, as well as in system cleanings between samples. Thus, it is impossible to predict how many patient samples a user will get from a reagent pack. As a result, the user must maintain an excess amount of reagents on hand, leading inevitably to a substantial degree of reagent waste. In addition, the hydraulics of these instruments, though generally less complex than the high-end laboratory systems, are still prone to the same reliability issues associated with the use of high numbers of vacuum lines, valves, and vacuum pumps, as well as with the small dimensions of the apertures used to count cells. Thus, for in-clinic and in-office applications, such low-end impedance instruments are severely deficient in producing the results needed for complete hematology analyses, are inefficient in their use of reagents, and have the same inherent reliability problems that are associated with high-end biological fluid analysis systems.

Some attempts have been made to address the clinical blood sample analysis needs of small laboratories and doctor's offices, but these efforts have met with only limited success. For example, the QBC.RTM. technology, developed by Becton-Dickinson, does not require flow cytometry or other expensive technology. The major moving part required is a centrifuge that forces the cellular components of blood to layer in an oversized, precisely bored capillary tube. Inside the capillary tube, a plastic "float" that is precisely manufactured for size and density characteristics establishes density equilibrium within the white blood cell and platelet layers, thereby expanding the height of the layers by about ten fold. This system provides fairly accurate estimates of platelet count, WBC count, and a two-part WBC differential (granular and agranular leukocytes), as well as a very accurate measurement of hematocrit. The QBC.RTM. 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.

While the QBC.RTM. technology addresses concerns of reliability and reagent waste for the in-clinic user, it falls well short of delivering the required parameters for a CBC, produces an incomplete differential, and does not quantify reticulocytes. Additionally, the precision and accuracy of the parameters produced by the QBC.RTM. system, except for hematocrit, are worse than those of the low-end impedance systems. Consequently, there remains a great need for an inexpensive and reliable biological fluid analysis system that is capable of producing the parameters that are relied upon by clinicians and doctors in making decisions that affect the treatment of a large percentage of their patients.

To address the need for such an instrument capable of providing hematology parameters that cannot be provided by low-end impedance instruments in an in-clinic setting, several major factors have to be considered. The size, cost and complexity of subsystems associated with high-end hematology systems must be reduced, while retaining the ability of the system to perform high-throughput blood analyses.

The major sub-systems that contribute to the cost and complexity of the high-end flow cytometry-based hematology systems are the light source, the optics system, and the fluid handling system. The gas lasers conventionally employed in flow cytometer-based hematology systems produce strong light signals, but high levels of noise associated with these lasers have minimized their utility for use in flow cytometry-based hematology systems. 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. However, this results in greater cost for the system. Incorporation of a laser diode in place of a conventional gas laser significantly reduces the total package size and cost. Diode lasers have inherently lower noise levels than gas lasers. However, the optical characteristics of laser diodes are not as desirable as those of gas lasers. Laser diodes are astigmatic, and have temperature-dependent and current-dependent noise regions, which causes a phenomenon 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 light measurement is very low, making it useless for measuring small particles (i.e., platelets), or to distinguish subtle difference in larger ones.

Precise temperature control and current control may be implemented to reduce mode hopping. However, this process requires that each laser diode be individually characterized to find a quiet temperature/current area where mode-hopping does not occur. Laborious effort is required to characterize each individual laser diode to find 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 is required. Furthermore, even if the ideal external temperature is determined, maintaining a constant temperature is complicated by the significant amount of heat produced when the diode laser is first turned on. As a result, temperature equilibrium of the diode laser can take up to 30 minutes to establish, even if the diode laser and temperature control system are working properly. This equilibration time decreases the useful life of the laser. Lastly, the implementation of temperature control severely limits the operating temperatures at which a unit can be used, and adds significant cost and size to a relatively inexpensive and small laser diode.

Significant costs and complexity in the optics system of flow cytometry-based hematology instruments are associated with the light beam shaping optics and the light collection optics. Beam shaping optics are used to alter the power distribution of a laser beam from a circular gaussian distribution to an ellipsoid distribution, where the power distribution is both more intense and more evenly distributed over the area of interest. Many hematology instruments use a collimated approach, where the "tails" of the gaussian power distribution curve are blocked, e.g., by an aperture. This creates a top hat effect, which provides uniformity over the region of interest and laser pointing stability while reducing the need for fluidic control. However, this approach reduces power density at the interrogation point in the flow cell.

Light beam shaping optics are also involved in the creation of a very thin light beam, sub-cellular in size (i.e., <4 microns), which is useful for time-of-flight (TOF) measurements and for maximizing power density at the point of cell interrogation. TOF measurements can be useful to distinguish bioparticles based on their sizes. Conventionally, the initial laser beam is first expanded, then focused to a small spot size. Both the expansion step and the focusing step are conventionally accomplished by using various optical elements, such as lenses, beamsplitters, apertures, and mirrors. Significant costs are incurred by the need to incorporate many expensive optical components. In addition to the high cost of each of these components, each optical component also must be held securely in space, and carefully aligned in three-dimensional space with the other components of the optic system. These steps add additional complexity and cost to the overall design.

Light collection optic systems in flow cytometry-based hematology systems are even more complex than the light 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 collect and steer scattered light 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.

Conventional hematology systems that automatically create a dilution of a whole blood sample generally do so in at least two separate steps. These two steps may be performed either serially or in parallel. Whether performed serially or in parallel, a minimum of two reaction chambers are utilized. One chamber may be used for counting and sizing of RBCs, while the other chamber may be used for counting and classifying WBCs. In some instruments, an additional chamber is added, for example to perform analyses of hemoglobin concentration. The reaction chambers used in conventional hematology systems are usually contained within the instrument, and must be rinsed between samples to prevent sample carryover.

In order to perform these tasks in the reaction chambers, a multitude of fluid handling components are required. These components include, for example, pressure pumps, to move diluent or cleaning solution around the system; vacuum pumps, to remove fluid from the reaction chambers; valves, to control the movement of fluids; metering devices, to precisely aliquot sample and reagents to the reaction chambers; and tubing to connect all the fluid handling components. The sheer number of components involved in the conventional hematology systems add greatly to the cost, and reduce the reliability, of such devices.

SUMMARY OF THE INVENTION

The device of the present invention is a flow-cytometry based bioparticle analysis system that combines unconventional flow cytometry light scatter measurement techniques with a unique approach to reagent delivery and sample processing that minimizes reagent waste and reduces systems and operational costs. More particularly, the present invention is directed to a flow cytometry-based hematology 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 be used in the analysis of whole blood samples, or in blood-derived samples, such as, for example, platelet-rich plasma, packed red blood cells, and the like. The system may also be used in the analysis of any biological sample other than blood or blood-derived products (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.

This present invention is a flow cytometry-based hematology system 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 use of a photodetector array that collects multiple light measurements without the need for any moving parts, lenses or other types of optical components greatly increases the reliability of the system. The photodetector array is preferably such that various types of scattered light are detectable, and the data collected provides a complete hematological analysis when used in combination with a high numerical aperture technique for right angle scatter. The laser light source may be a standard diode laser light source that is shaped to produce a thin line, as disclosed in copending U.S. patent application Ser. No. 09/488,211, filed on Jan. 19, 2000. The diode laser reduces the size and complexity of the system. Mode-hopping, a problem that occurs with diode lasers, may be reduced significantly by the application of a high-frequency modulation technique. Finally, the system preferably uses a unique system of consumable reagent tubes that act as disposable reaction chambers, mixing chambers, and waste chambers for the blood sample analyses. The consumable tubes incorporate polystyrene latex particles or the like (i.e., 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, greater reliability, and greatly improved accuracy and validation of the results.

Accordingly, one object of the present invention is to provide a lensless light detection system for a flow cytometer.

It is another object of the present invention to provide a lensless light detection system that comprises a photodetector array on an integrated circuit chip which is effective to measure at least one of axial light loss, low-angle forward scattered light, and high-angle forward scattered light.

It is another object of the present invention to provide a lensless light detection system that comprises a photodetector array, arranged on a plurality of electrically distinct integrated circuit chips, which is effective to measure at least one of axial light loss, low-angle forward scattered light, and high-angle forward scattered light.

It is another object of the present invention to provide a disposable vessel for use in a flow cytometry-based hematology system that comprises a tube; a known number of reference particles in the tube, each of the reference particles having a predetermined diameter such that, when the reference particles are illuminated by light, the light is scattered by the reference particles such that the scattered light falls into at least one of a plurality of predetermined light scatter channels; and at least one reagent.

It is another object of the present invention to provide a disposable vessel for use in a flow cytometry-based hematology system that comprises a tube; a known number of reference particles in the tube, each of the reference particles having one of several diameters such that, when the reference particles are illuminated by light from a laser, the light is scattered by the reference particles such that the scattered light falls into one of a plurality of predetermined light scatter channels; and at least one reagent.

It is another object of the present invention to provide a method for analyzing a blood sample or a blood-derived sample using a flow cytometry-based hematology system that comprises mixing the blood sample or blood-derived sample with a reagent in a disposable vessel containing a known number of reference particles, the reference particles having one of several diameters such that when the reference particles are illuminated by light, the light is scattered such that the scattered light falls into one of a plurality of predetermined light scatter channels; standardizing the sample using the reference particles in the disposable vessel containing the portion by measuring at least one of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight for each of the reference particles, and comparing the measurements to predetermined values obtained for the reference particles; counting cells from the sample; measuring at least one parameter from the group consisting of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight for each cell in the sample, to produce a measurement value for each parameter; and analyzing the measurement values to classify each cell.

It is another object of the present invention to provide a method for analyzing a blood sample or a blood-derived sample using a flow cytometry-based hematology system that comprises mixing a first portion of the blood sample or blood-derived sample with a reagent in a disposable vessel containing a known number of reference particles to form a red cell solution, the reference particles having one of several diameters such that when the reference particles are illuminated by light, the light is scattered such that the scattered light falls into one of a plurality of predetermined light scatter channels; standardizing the red cell solution and the flow cytometry-based hematology system using the reference particles in the disposable vessel containing the portion by measuring at least one of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight for each of the reference particles, and comparing the measurements to predetermined values obtained for the reference particles; counting red blood cells, reticulocytes, and platelets from the red cell solution; measuring at least one parameter from the group consisting of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight for each cell in the red cell solution, to produce a red cell solution measurement value for each parameter for each of the red blood cells, reticulocytes, and platelets from the sample; mixing a second portion of the blood sample or blood-derived sample with a lyse solution in the disposable vessel containing a known number of reference particles to form a white cell solution; counting white blood cells from the white cell solution; measuring at least one parameter from the group consisting of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight for each cell in the white cell solution, to produce a white cell solution measurement value for each parameter for the white blood cells; analyzing the red cell solution measurement values and the white cell solution measurement values to classify each cell.

It is another object of the present invention to provide a method for analyzing a blood sample or a blood-derived sample using a flow cytometry-based hematology system that comprises mixing the blood sample or blood-derived sample with a reagent in a disposable vessel containing a known number of reference particles, the reference particles having one of several diameters such that when the reference particles are illuminated by light, the light is scattered such that the scattered light falls into one of a plurality of predetermined light scatter channels; standardizing the sample using the reference particles in the disposable vessel containing the portion by measuring at least one of axial light loss, low-angle forward scattered light, high-angle forward scattered light, right angle scattered light, and time-of-flight for each of the reference particles, and comparing the measurements to predetermined values obtained for the reference particles; counting cells from the sample; measuring at least one parameter for the sample by using one of a platelet aggregation assay and a clottable assay, to produce a measurement value for each parameter; and analyzing the measurement values.

It is another object of the present invention to provide a flow cytometry-based hematology system for classifying and counting biological cells in a blood sample or blood-derived sample that comprises a disposable vessel, the disposable vessel containing a known number of reference particles and at least one reagent, the reference particles having one of several diameters such that when the reference particles are illuminated by light, the light is scattered such that the scattered light falls into at least one of a plurality of known light scatter channels; a lyse tube, the lyse tube containing at least one lyse reagent capable of lysing red blood cells that may be present in the blood sample or blood-derived sample while leaving white blood cells in the blood sample or blood-derived sample substantially intact for a period of at least one minute; a sheath tube, the sheath tube containing a sheath solution capable of acting as a sample dilution reagent, a sheath reagent, and a washing reagent for a hydraulic path of the flow cytometry-based hematology system; a flow cell; a hydraulic system, the hydraulic system being capable of mixing the blood sample or blood-derived sample with one or more of the contents of the disposable vessel, the lyse tube and the sheath tube, and being capable of moving one or more of the disposable vessel, the lyse tube, the sheath tube, or a mixture of the blood sample or the blood-derived sample with one or more of the contents of the disposable vessel, the lyse tube and the sheath tube through the flow cell; a diode laser, the diode laser emitting light to irradiate cells derived from the blood sample or the blood-derived sample in the flow cell; a lensless light detection system, the lensless light detection system having a first portion capable of collecting one or more of axial light loss, low-angle scattered light and high-angle scattered light scattered by the cells in the flow cell when the cells in the flow cell are irradiated by the laser, and a second portion capable of collecting right angle scattered light scattered at a high numerical aperture, the second portion being physically distinct from the first portion, the first portion and the second portion converting the axial light loss, the low-angle scattered light, the high-angle scattered light, and the right angle scattered light into electrical signals; and a signal processor, the signal processor being capable of analyzing the electrical signals generated from the first portion and the second portion of the lensless optical module, and generating data therefrom.

It is another object of the present invention to provide a flow cytometry-based hematology system for classifying and counting biological cells in a blood sample or blood-derived sample that comprises a disposable vessel, the disposable vessel containing a known number of reference particles and at least one reagent, the reference particles having one of several diameters such that when the reference particles are illuminated by light, the light is scattered such that the scattered light falls into one of a plurality of predetermined light scatter channels; a lyse tube, the lyse tube containing at least one lyse reagent capable of lysing red blood cells that may be present in the blood sample and blood-derived sample while leaving white blood cells in the sample substantially intact for a period of at least one minute; a sheath tube, the sheath tube containing a solution capable of acting as a sample dilution reagent, a sheath reagent, and a washing reagent for a hydraulic path of the flow cytometry-based hematology system; a flow cell; a hydraulic system, the hydraulic system being capable of mixing the blood sample or blood-derived sample with one or more of the contents of the disposable vessel, the lyse tube, and the sheath tube, and being capable of moving one or more of the contents of the disposable vessel, the lyse tube, the sheath tube, or a mixture of the blood sample of the blood-derived sample with one or more of the contents of the disposable vessel, the lyse tube, and the sheath tube through the flow cell; a diode laser, the diode laser emitting light to irradiate cells from the blood or blood-derived sample in the flow cell; a lensless light detection system, the lensless light detection system having a first portion capable of collecting axial light loss, low-angle forward scattered light, and high-angle forward scattered light, and a second portion capable of collecting right angle scattered light scattered at a high numerical r aperture; and 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 light detection system, and generating data therefrom.

The above and other objects, features and advantages of the present invention will become apparent from the following description read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the system hydraulics of a preferred embodiment of the present invention.

FIG. 2 is a drawing of a flow cell assembly used in an optical detection system of a preferred embodiment of the present invention.

FIG. 3A is a top view schematic representation of a laser beam shaping optic system of a preferred embodiment of the present invention.

FIG. 3B is a side view schematic representation of a laser beam shaping optic system of a preferred embodiment of the present invention.

FIG. 4 is an illustration of an optic system of a preferred embodiment of the present invention, showing a light source, a flow cell, a photodetector array, and a right-angle scatter detector.

FIG. 5 is a schematic illustration of a Mie mask used in a photodetector of a preferred embodiment of the present invention.

FIG. 6 is a schematic representation of the events that occur in a consumable tube during the system cycle of a preferred embodiment of the device of the present invention.

FIG. 7 is a schematic illustration of an embodiment of a tube positioning assembly used in a preferred embodiment of the present invention.

FIG. 8 is a schematic illustration of a mixing and piercing assembly of a preferred embodiment of the present invention.

FIG. 9 is a schematic illustration of the system hydraulics of a preferred embodiment of the present invention.

FIG. 10A is a histogram of digitized data collected from an unlysed whole blood sample, in the presence of added latex particles, by a preferred embodiment of the present invention. Low-angle forward scatter channel (FSL) data is shown on the x-axis, and the number of events (or occurrences) collected for each of the individual channels are shown on the y-axis. A=platelet events. B=latex particle events. C=red blood cell events.

FIG. 10B shows data collected from the same sample as in FIG. 10A, except FIG. 10B depicts the extinction or axial light loss channel (EXT) data on the x-axis.

FIG. 10C shows data collected from the same sample as in FIG. 10A, except FIG. 10C depicts the right angle scatter channel (RAS) data on the x-axis.

FIG. 10D shows data collected from the same sample as in FIG. 10A, except FIG. 10C depicts the time-of-flight (TOF) or pulse width measurement on the x-axis. Pulse width measurements can be obtained from any of the channels that utilize a photodetector, but it is preferred to use either the EXT channel or the FSL channel.

FIG. 11A displays the red cell, platelet and latex particle data from FIGS. 10B and 10C, in scatter-plot form. Scatter plots are a representation of different data collected simultaneously from a cell or particle. Data collected from one particle is plotted on the x-axis against data from another particle on the y-axis.

FIG. 11B displays the red cell, platelet and latex particle data from FIGS. 10A and 10C, in scatter-plot form.

FIG. 12A is a histogram of digitized data collected from a lysed whole blood sample, in the absence of added latex particles, by a preferred embodiment of the present invention. The extinction (EXT) channel data are shown on the x-axis, and the number of events (or occurrences) collected for each of the individual channels are shown on the y-axis. A=lymphocytes; B=monocytes; and C=granulocytes.

FIG. 12B is a histogram of digitized data collected from a lysed whole blood sample, in the presence of added latex particles, by a preferred embodiment of the present invention. The extinction (EXT) channel data are shown on the x-axis, and the number of events (or occurrences) collected for each of the individual channels are shown on the y-axis. A=lymphocytes; B=monocytes; C=granulocytes; and D=latex particles.

FIG. 12C is a histogram of digitized data collected from a lysed whole blood sample, in the absence of added latex particles, by a preferred embodiment of the present invention. The forward low scattered light (FSL) channel data are shown on the x-axis, and the number of events (or occurrences) collected for each of the individual channels are shown on the y-axis. A=lymphocytes; B=monocytes; and C=granulocytes.

FIG. 12D is a histogram of digitized data collected from a lysed whole blood sample, in the presence of added latex particles, by a preferred embodiment of the present invention. The forward low scattered light (FSL) channel data are shown on the x-axis, and the number of events (or occurrences) collected for each of the individual channels are shown on the y-axis. A=lymphocytes; B=monocytes; C=granulocytes; and D=latex particles.

FIG. 12E is a histogram of digitized data collected from a lysed whole blood sample, in the absence of added latex particles, by a preferred embodiment of the present invention. The right angle scattered light (RAS) channel data are shown on the x-axis, and the number of events (or occurrences) collected for each of the individual channels are shown on the y-axis. A=lymphocytes; B=monocytes; and C=granulocytes.

FIG. 12F is a histogram of digitized data collected from a lysed whole blood sample, in the presence of added latex parti