Idexx Laboratories, Inc.

Ortho Diagnostic Systems Inc.

Ortho Diagnostics, Inc.

Sienna Biotech, Inc.

Union Biometrica, Inc.

Other

Point Care Technologies

Union Biometrica Technology Holdings, Inc.

W Peter Hansen

Petra B Krauledat

John W Roche

Igino Lombardo

Russell J Gershman

Robert A Hoffman

Anthony A Ferrante

Harold C Flynn, Jr

Michael Cennerazzo

Donald Francis Perrault, Jr

Peter J Natale

Donald E Barry

Harold Richardson Crews

J Garland O'Connell

George B Hovorka

Peter B Kramer

Manish S Kochar

Carl Theodore Edens

Michelle L Coleman

Marcus F Julian

Alan M Hochberg

James W Russell

Harold C Flynn

This invention relates to the field of biological assays where cells can be classified and enumerated using flow cytometry optical instrumentation. The invention combines information from multi-angle, light scatter from the cell itself and multi-angle light scatter from small, optically resonant particles that are selectively bound to surface molecules on the cell to carry out classification and enumeration. This light scatter method enables an instrumentation system that is simple to use, inexpensive to build, and mechanically robust; making it suitable for use in remote clinical environments.

Enhanced cellular assay method for use in flow cytometry or similar instruments using optically resonant particles

A method of using elongate multicellular organisms in conjunction with a specialized flow cytometer for drug discovery and compound screening. A stable, optically detectable linear marker pattern on each organism is used to construct a longitudinal map of each organism as it passes through the analysis region of the flow cytometer. This pattern is used to limit complex data analysis to particular regions of each organism thereby simplifying and speeding analysis. The longitudinal marker pattern can be used to alter signal detection modes at known regions of the organism to enhance sensitivity and overall detection effectiveness. A repeating pattern can also be used to add a synchronous element to data analysis. The marker patterns are established using known methods of molecular biology to express various indicator molecules. Inherent features of the organism can be rendered detectable to serve as marker patterns.

System for axial pattern analysis of multicellular organisms

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.

Consumable tube for use with a flow cytometry-based hematology system

Flow cytometry-based hematology system

An instrument for analyzing and dispensing objects larger than about 70 &mgr;m in diameter is based on a flow cytometer with a novel fluidic switch arrangement for diverting a portion of a sample stream in response to detector signals in a flow cell. The instrument is particularly adapted for dispensing multicellular test organisms like nematodes or large microspheres for use in screening large libraries of potential pharmaceutical agents. Hydrodynamic focussing is used to center and align the objects in the flow cell. The objects pass through a sensing zone where optical or other characteristics of the objects are detected. The detector signals are processed and used to operate a fluidic switch that is located downstream from the sensing zone. The fluid stream containing the detected objects emerges from the flow cell into air where a fluid stream controlled by the fluidic switch diverts portions of the stream containing no sample objects or sample objects not meeting predetermined characteristics. The undiverted sample stream deposits selected sample objects into a plurality of containers.

Instrument for selecting and depositing multicellular organisms and other large objects

The high numerical aperture flow cytometer of the present invention includes a flow cell and a laser input. The laser input emits a beam of light that is oriented substantially orthogonally to the flow of blood cells through the flow cell such that laser light impinges upon the blood cells as they pass through the flow cell. A portion of the beam from the laser input that impinges upon the blood cells in the flow cell is scattered at a substantially right angle to the beam of laser input (&ldquo;right angle scatter&rdquo;). A second portion of the beam from the laser input that impinges upon the cells in the flow cell is scattered at a much lower angle than 90&deg;. This scatter is termed forward scatter light&rdquo; and is collected on two distinct photo detectors, that represent 'forward scatter low' (FSL) which has an angle of from about 1&deg; to about 3&deg;, and 'forward scatter high' (FSH) which has an angle of from about 9&deg; to about 12&deg; from the orientation of the original beam from laser input. A third photo detector is placed in between these two forward scatter detectors, that is axial with the impinging laser light. This detector measures axial light loss, or light extinction (EXT) which is the sum of all the light that is absorbed and scattered by the blood cells. A right angle scatter light detector is oriented to receive the previously mentioned right angle scatter light. A forward scatter light detector is oriented to capture the previously mentioned forward scatter light oriented different angles from the beam of the laser input.

High numerical aperture flow cytometer and method of using same

A device and a method enable the rapid, quantitative evaluation of a large collection of ligands for binding affinity with a certain immobilized receptor, the improvements being that binding pan be detected without the need for a label and that binding is carried out in solution phase at a high rate. The instrument has at least two embodiments, one is based on a sensitive absorption photometer and the other on a sensitive light scatter photometer operating at a specific resonance wavelength, &lgr; , of small, metallic, colloidal particles. The resonance is present in small particles having a complex refractive index with real part n(&lgr;) approaching 0 and imaginary part k(&lgr;) approaching 2 simultaneously at a specific wavelength &lgr; . The particles are substantially spherical and substantially smaller than &lgr; . The receptor is immobilized on a suspension of such particles and ligand binding is detected by a change in optical absorption or light scatter at the resonance wavelength.

Binding assays using optical resonance of colloidal particles

The high numerical aperture flow cytometer of the present invention includes a flow cell and a laser input. The laser input emits a beam of light that is oriented substantially orthoganilly to the flow of blood cells through the flow cell such that laser light impinges upon the blood cells as they pass through the flow cell. A portion of the beam from the laser input that impinges upon the blood cells in the flow cell is scattered at a substantially right angle to the beam of laser input (&ldquo;right angle scatter&rdquo;). A second portion of the beam from the laser input that impinges upon the cells in the flow cell is scattered at a much lower angle than 90&deg;. This scatter is termed &ldquo;low angle forward scatter light&rdquo; and has an angle of from about 2&deg; to about 5&deg; from the orientation of the original beam from laser input. A right angle scatter light detector is oriented to receive the previously mentioned right angle scatter light. A low angle forward scatter light detector is oriented to capture the previously mentioned low angled forward scatter light oriented at about 2&deg; to about 5&deg; from the beam of the laser input.

Disclosed are an optical flow particle apparatus and method for conducting a particle light scatter-based immunoassay for simultaneously measuring the presence or amount of one or more analytes in a fluid sample which involves the use of a reagent set for each analyte including first binding molecule-coated monodisperse microspheres and second binding molecule-coated colloidal particles in which at least one of the first or second binding molecules specifically binds a respective one of the analytes. In the case where more than one analytes are detected, each monodisperse microperse microsphere of a particular reagent set has a light scatter signal resolvable from that of microspheres of any other reagent set. Changes determined in the distributions of the measured light scatter signals for individual microspheres of each of the particular reagent sets are indicative of the presence or amount of the respective analyte(s) in the sample.

Light scatter-based immunoassay

A homogeneous method of measuring chemical binding relies on resonant, or 'amplified,' optical extinction (light scattering plus absorption) from a defined, specific class of colloidal particles wherein the real term n of the complex refractive index n-ik approaches zero while the imaginary term k approaches .sqroot.2. Chemical binding partners are coated onto the particles, which either aggregate or disperse during the binding reaction, causing an optical extinction change at one wavelength that is quantitatively related to the number of single colloidal particles and another at a second wavelength that is quantitatively related to the number of doublet colloidal particles. The method employs: (1) a specific class of colloidal particles that exhibit optical resonance and that are substantially smaller in diameter than a wavelength of visible light; (2) a one-step process of colloidal particle aggregation; and (3) a photometric extinction measurement at wavelengths where the particles exhibit optical resonance.

Homogeneous binding assay

A homogeneous immunoassay method for the simultaneous determination of one or more antibody, antigen or hapten analytes in a fluid sample, that comprises the quantification of the effect of said analytes on the statistical changes in a dimension of a light scatter pulse height distribution histogram of relatively large diameter monodisperse binding molecule-coated polymeric microspheres induced by the binding to said microspheres of polydisperse binding molecule-coated colloid metal particles of relatively small diameter. For simultaneous assays of multiple analytes, different diameter or refractive index microspheres are assigned to each analyte. The assay may be used in forward binding, displacement, inhibition, and competition type systems, with the direction of the change in histogram dimension depending on the system. A convenient dimension to measure is the normalized peak width of a graphical representation of the histogram. For simultaneous assays for multiple analytes, a monodisperse polymeric microsphere of unique diameter or refractive index is dedicated to each analyte, so as to generate multiple histograms, one for each analyte. Polydisperse binding molecule-coated colloid metal particles, when used in effective concentrations, will also serve as a scavenger means to reduce the interfering effects of non-specific substances in the analyte-containing fluid samples, particularly when said samples are of biological origin.

Light scatter-based immunoassay without particle self aggregation

A particle agglutination-based, stable kinetic method for simultaneously determining the concentrations of multiple analytes in a single fluid sample with the addition of a single reagent, that entails the use of a novel high resolution sheath flow cell, a novel optical flow particle analyzer (FPA), and unidirectional low angle forward light scattering from multiply-sized or refractive indexed, differently coated particles and their aggregates.

Simultaneous multiple assays

A particle agglutination-based, stable kinetic method for simultaneously determining the concentrations of multiple analytes in a single fluid sample with the addition of a single reagent, that entails the use of a novel high resolution sheath flow cell, a novel optical flow particle analyzer (FPA), and unidirectional low angle forward light scattering from multiply-sized or refractive indexed, differently coated particles and their aggregates. Also disclosed are two embodiments of an instrument specifically designed to carry out the method of the invention.

Methods for attaching one component of a ligand-anti-ligand pair onto a solid phase surface. The component may be attached to a photoactivatable cross-linker capable of coupling the component to a colloidal medium coating the surface or alternatively, the component may be coupled to a bead and held in place against the surface by an overlay of the colloidal media having voids and spaces smaller than the diameter of the particle but large enough to permit diffusion of the other of the ligand-anti-ligand pair to be detected.

Growing community of inventors

Canaan, NY, United States of America

W Peter Hansen