Abstract: Disclosed are apparatus and methods for analyzing bodily fluids, such as blood samples, using an integrated hematology analyzer and flow cytometer system. Under the present approach, an integrated system may operate as a closed fluidic system or an open fluidic system, and may selectively perform automated hematologic protocols, flow cytometer protocols, and custom protocols. Such apparatus may, for example, identify and enumerate multiple cell types in whole blood based on cellular morphology, analyze cellular immunoassays using antibodies labeled to cells, and also detect low abundant analytes in whole blood as well as serum and other bodily fluids not attached to cells using bead-based immunoassay methods. The system may include a fluid handling system to control sample flow, an optical transducer that includes a flow cell, optical detectors for light scatter and/or fluorescence, and also an illumination source.

Abstract: Disclosed herein are apparatus, systems, and methods for optically identifying and enumerating cells present in a blood sample. A light scatter detector array may be used having no more than three light scatter detectors. The array may include a side scatter detector, an intermediate angle light scatter detector, and one of an axial light loss detector and a forward light scatter detector. A lytic reagent system is disclosed that allows for the identification and enumeration of five major leukocyte populations in normal whole blood on an instrument using no more than three light scatter detectors.

Abstract: Disclosed herein are apparatus, systems, and methods for optically identifying and enumerating cells present in a blood sample. A light scatter detector array may be used having no more than three light scatter detectors. The array may include a side scatter detector, an intermediate angle light scatter detector, and one of an axial light loss detector and a forward light scatter detector. A lytic reagent system is disclosed that allows for the identification and enumeration of five major leukocyte populations in normal whole blood on an instrument using no more than three light scatter detectors.

Abstract: Disclosed are apparatus and methods for analyzing bodily fluids, such as blood samples, using an integrated hematology analyzer and flow cytometer system. Under the present approach, an integrated system may operate as a closed fluidic system or an open fluidic system, and may selectively perform automated hematologic protocols, flow cytometer protocols, and custom protocols. Such apparatus may, for example, identify and enumerate multiple cell types in whole blood based on cellular morphology, analyze cellular immunoassays using antibodies labeled to cells, and also detect low abundant analytes in whole blood as well as serum and other bodily fluids not attached to cells using bead-based immunoassay methods. The system may include a fluid handling system to control sample flow, an optical transducer that includes a flow cell, optical detectors for light scatter and/or fluorescence, and also an illumination source.

Abstract: The present disclosure describes a method of monitoring disease progression in a mammal positive for immunodeficiency virus which includes collecting blood cells from a mammal to obtain a first blood sample adding antibodies such as CD4 and CD8 to the first blood sample scanning the blood sample to produce a first multivariate dot plot which may be used to quantify at least CD4+ and CD8+ blood cell populations to produce a first ratio. The first multivariate dot plot may also be used to quantify a CD8??low subpopulation which may be used to calculate a second ratio. A third ratio is calculated of the second ratio to the first ratio and the result plotted on a graph as a first point. This process may be repeated to produce a second point for evaluating an extent of disease progression.

Abstract: A lytic reagent composition and methods for differential analysis of leukocytes are disclosed. In embodiments, the analysis may utilize optical measurements in flow cytometry based hematology analyzers. The reagent system includes an anionic surfactant in a hypotonic solution, an inorganic buffer to maintain the pH in a range from about 6 to about 10, and optionally a leukocyte stabilizer. The reagent system is used to lyse red blood cells and stabilize the leukocytes to enable multi-part differentiation of leukocytes in a near physiologic pH environment on a flow cytometry based hematology analyzer using axial light loss, light scatter intensity, high-numerical aperture side scatter, and time-of-flight measurements.

Abstract: A lytic reagent composition and methods for differential analysis of leukocytes are disclosed. In embodiments, the analysis may utilize optical measurements in flow cytometry based hematology analyzers. The reagent system includes an anionic surfactant in a hypotonic solution, an inorganic buffer to maintain the pH in a range from about 6 to about 10, and optionally a leukocyte stabilizer. The reagent system is used to lyse red blood cells and stabilize the leukocytes to enable multi-part differentiation of leukocytes in a near physiologic pH environment on a flow cytometry based hematology analyzer using axial light loss, light scatter intensity, high-numerical aperture side scatter, and time-of-flight measurements.

Abstract: An apparatus for use in controlling the temperature of one or more substances passing through one or more microfluidics channels in an analysis device is set forth. The apparatus includes a heating unit having first and second surfaces. The first surface of the heating unit is constructed so that it is at least partially exposed for cooling of the heating unit. The apparatus also includes a thermally conductive medium that is disposed proximate the second surface of the heating unit. The one or more microfluidics channels are disposed in the thermally conductive medium. In one embodiment, the one or more microfluidics channels are in the form of a plurality of capillary columns, such as those used in instruments for capillary electrophoresis. Each capillary column is substantially surrounded by the material forming the thermally conductive medium.

Abstract: An apparatus for use in controlling the temperature of one or more substances passing through one or more microfluidics channels in an analysis device is set forth. The apparatus comprises a heating unit having first and second surfaces. The first surface of the heating unit is constructed so that it is at least partially exposed for cooling of the heating unit. The apparatus also comprises a thermally conductive medium that is disposed proximate the second surface of the heating unit. The one or more microfluidics channels are disposed in the thermally conductive medium. In one embodiment, the one or more microfluidics channels are in the form of a plurality of capillary columns, such as those used in instruments for capillary electrophoresis. Each capillary columns is substantially surrounded by the material forming the thermally conductive medium.

Abstract: A thermostat control system, that can be configured to include an array of two or more capillary columns or two or more channels in a microfabricated device, is disclosed. A thermally conductive material is in contact with each column or channel in the array. One or more independently controlled heating or cooling elements is positioned adjacent to or within the thermally conductive material, each heating or cooling element being connected to a source of heating or cooling. One or more independently controlled temperature sensing elements and one or more independently controlled temperature probes are also positioned adjacent to or within the thermally conductive material. Each temperature sensing element is connected to a temperature controller, and each temperature probe is connected to a thermometer.

Abstract: The dyes of the present invention are useful for many purposes that include markers or tags for detecting the presence of a molecule or compound to which they are bound. The dyes may be either red-excitable or blue-excitable. The dyes of the invention are particularly well suited for staining of nucleic acids. For example, these dyes are particularly suitable for staining of RNA in reticulocytes. In another exemplary application, these dyes are suitable for staining DNA in nucleated red blood cells. Typically, when used in staining of nucleic acids, the dyes are formulated into reagent solutions. In addition, the invention provides compositions and methods for facilitating rapid transport of dye molecules through a cell membrane. Such rapid staining requires that a sample be contacted with a dye composition of the invention in the presence of at least one surfactant and optionally, a sulfonic acid or a salt thereof.

Abstract: A dye compound having the formula I: wherein, n is 0, 1, 2, or 3; R1 is H, alkyl, or an alkoxy group; R2 is CH2(CH2)mOH, wherein m is 0, 1, 2, or 3; X is O, S, or C(CH3)2; R is CH3, CH(CH3)2, CH2CH2OH, alkyl, alkylsulfonate, or hydroxyalkyl and B− is a counteranion. Red- and blue-excitable dyes based on the above chemical structure are described, and reagents containing them are described for use in staining nucleic acid, more particularly for staining reticulocytes. Also described are methods for detecting reticulocytes using such compositions.

Abstract: The dyes of the present invention are useful for many purposes that include markers or tags for detecting the presence of a molecule or compound to which they are bound. The dyes may be either red-excitable or blue-excitable. The dyes of the invention are particularly well suited for staining of nucleic acids. For example, these dyes are particularly suitable for staining of RNA in reticulocytes. In another exemplary application, these dyes are suitable for staining DNA in nucleated red blood cells. Typically, when used in staining of nucleic acids, the dyes are formulated into reagent solutions. In addition, the invention provides compositions and methods for facilitating rapid transport of dye molecules through a cell membrane. Such rapid staining requires that a sample be contacted with a dye composition of the invention in the presence of at least one surfactant and optionally, a sulfonic acid or a salt thereof.

Abstract: A rapid fluorescence staining method for facilitating flow cytometry analysis of reticulocytes is described. The method comprises contacting cells with a cocktail containing a detergent, sphering agent, and a cell impermeable dye, such as TO-PRO-3, for about one minute. Advantageously, the inventors have found that the cocktail permits the dye to penetrate the cell membrane rapidly.

Abstract: A method and apparatus for differentiating and enumerating the five major sub-populations of leukocytes in a blood sample (i.e., lymphocytes, monocytes, eosinophils, neutrophils and basophils) uses multiangle light scatter and DC (Coulter) volume measurements. Light scattering characteristics of the leukocytes are determined within five different angular ranges, all being lower than 40 degrees. The invention is particularly useful in differentiating and enumerating the basophil sub-population which has heretofore required more complex apparatus and/or chemical processing.

Abstract: A method for enumerating and distinguishing between blood cell populations in a biological sample includes the steps of contacting a biological sample with a cell membrane-permeant, red-excited, nucleic acid binding dye, without significantly disrupting the integrity of the cells; exciting this sample with light in one red wavelength; and measuring fluorescence emitted from different cell populations in the sample. This method is particularly desirable for enumerating different WBC subpopulations using flow cytometry. This method is also useful for enumerating reticulocytes or NRBC or mature RBC. This method is enhanced by pretreating the sample with a nucleic acid-specific blocking agent. Dyes useful in this method include, without limitation, SYTO®17 dye, SYTO®159 dye, SYTO®60 dye, SYTO®61 dye, SYTO®62 dye, SYTO®63 dye, and SYTO®64 dye.

Abstract: Time-resolved fluorescence decay measurements for flowing particles. An apparatus and method for the measurement and analysis of fluorescence for individual cells and particles in flow are described, wherein the rapid measurement capabilities of flow cytometry and the robust measurement and analysis procedures of time-domain fluorescence lifetime spectroscopy are combined. A pulse-modulated cw laser is employed for excitation of the particles. The characteristics and the repetition rate of the excitation pulses can be readily adjusted to accommodate for fluorescence decays having a wide range of lifetimes.