Becton Dickinson and Company Limited

Diether J Recktenwald

Thomas J Mercolino

Chia H Chen

Stephen C Wardlaw

Robert A Levine

Leon W Terstappen

Michael R Loken

Majid Mehrpouyan

Janette T Phi-Wilson

Rickie S Kerndt

Rudolf Varro

Edward M Goldberg

Hrair Kirakossian

Anthony J Ward

The present disclosure provides cytometric methods for the detection of rare target cells in a sample. In certain aspects, the methods and compositions may facilitate the detection of rare target cells, such as circulating tumor cells (CTCs), in a biological sample such as blood. Aspects of the methods include contacting the sample with first and second binding members that specifically bind to a marker of the rare target cell, and cytometrically assaying the sample for the presence of cells comprising bound first and second binding members to detect the rare target cell in the sample. Also provided are systems, compositions, and kits for practicing the subject methods.

Methods and compositions for cytometric detection of rare target cells in a sample

Arrays of microparticle populations, each population labeled with a single fluorescent dye, are provided for use in multiplex assays. The populations form a virtual multidimensional array wherein each microparticle is identified by fluorescence intensity in two different fluorescence detection channels. The arrays are useful in a variety of assays, including multiplex, multi-analyte assays for the simultaneous detection of two or more analytes by, for example, flow cytometry, and a labeling reagents in, for example, microscopy. The use of singly-dyed microparticles to form multidimensional arrays greatly simplifies the creation of multiplex assays.

Multiplex microparticle system

This invention comprises a one step method for the detection and enumeration of absolute counts of one or more cell populations in a blood sample. The method employs a reagent comprising a mixture of one or more cell markers, a fluorescent microparticle and a fixative. The reagent may be combined with unlysed whole blood and analyzed by means of flow cytometry.

One step method for detection and enumeration of absolute counts of one

A patient's health is diagnosed by centrifuging blood samples in a transparent tube, which tube contains one or more groups of particles such as lyposomes or plastic beads of different densities for each group. Each group of density-defined particles carries antigens or antibodies which are specific to a complement antigen or antibody which may be in the blood sample being tested, and which are indicative of the patient's health. A label-tagged antibody which is specific to all bound antibody/antigen couples is added to the blood sample so as to form labelled antibody+antigen-antibody complexes (AAAC) in the blood sample. Upon centrifugation, the complexed particles will settle out in different areas in the tube according to the respective density of the particles, and the degree of label emission of the particle layers can enable qualitative or quantitative analyses of the blood sample to be made. Unbound labelled antibodies will be washed away from the complexed layers by the washing action of the descending blood cells during the centrifugation step. Unbound labelled antibodies will thus not interfere with the analysis.

Indirect fluorescent assay of blood samples

A patient's health is diagnosed by centrifuging blood samples in a transparent tube, which tube contains one or more groups of particles such as lyposomes or plastic beads of different densities for each group. Each group of density-defined particles carries antigens or antibodies which are specific to a complement antigen or antibody which may be in the blood sample being tested, and which are indicative of the patient's health. A label-tagged antibody which is specific to all bound antibody/antigen couples is added to the blood sample so as to form labeled antibody+antigen-antibody complexes (AAAC) in the blood sample. Upon centrifugation, the complexed particles will settle out in different areas in the tube according to the respective density of the particles, and the degree of label emission of the particle layers can enable qualitative or quantitative analyses of the blood sample to be made. Unbound labeled antibodies will be washed away from the complexed layers by the washing action of the descending blood cells during the centrifugation step. Unbound labeled antibodies will thus not interfere with the analysis.

Apparatus for indirect fluorescent assay of blood samples

A method for improving recovery of cells from liquid suspension by centrifugation. The method comprises coating the interior of centrifuge containers with a solution comprising an amphipathic compound prior to introduction of the cell suspension and centrifugation. The method is particularly suited for recovery or concentration of rare cells from dilute suspensions, for example when rare cells are isolated from a sample by sorting on a flow cytometer.

Coating agents for cell recovery

A method for improving recovery of cells from liquid suspension by centrifuqation. The method comprises coating the interior of centrifuge containers with a solution comprising an amphipathic compound prior to introduction of the cell suspension and centrifugation. The method is suited for recovery or concentration of rare cells from dilute suspensions, for example when rare cells are isolated from a sample by sorting on a flow cytometer.

Peridinin-chlorophyll-protein complexes are provided for use as fluorescent labels and are particularly useful in diagnostic assays employing as a reagent a fluorescent compound conjugated to a member of a specific binding pair, wherein the pair consists of a biochemical ligand and a receptor and the diagnostic assay comprises a step in which the conjugate binds to its complementary binding-pair member.

Peridinin-chlorophyll complex as fluorescent label

A method for calibrating an instrument for using that instrument to obtain at least one light-related signal from particles under analysis comprises directing an incident beam of light at calibration particles having one or more known characteristics related to the particles expected to be analyzed. Both a light signal and a noise signal from the calibration particles are detected. A measurement is made of the ratio of the detected light signal to the detected noise signal, and that measurement is reported. The measured ratio is then compared to a predetermined ratio which represents a threshold for minimum instrument performance. This method further includes adjusting, if the predetermined ratio has not been attained, the operation of the instrument, while the calibration particles are within the incident beam of light, until the measured ratio reaches the predetermined ratio whereby the instrument is calibrated for subsequently obtaining the light signal from particles to be analyzed. A kit of reagents for use in calibrating an instrument for performing two-color analysis of particles and particle standars for calibrating fluorescence analysis instruments are also part of the instant invention.

Method and materials for calibrating flow cytometers and other analysis

A method for determining one or more characteristics of particles using multiple fluorescence analysis comprises directing an incident light beam at the particles under analysis. The particles include at least three fluorescent markers each having different emission spectra. The incident light beam causes the excitation of the markers by light at a single wavelength whereby different wavelengths of fluorescence are emitted from the particles. Different fluorescence emissions associated with the particles under analysis are simultaneously detected. This method further includes associating the detected fluorescence with one or more characteristics of the particles. An apparatus is also part of the present invention for carrying out the aforementioned method.

Multi-color fluorescence analysis with single wavelength excitation

A method for identifying and enumerating cells of a subclass of blood cells in relation to cells of another subclass of blood cells is provided. In the method a first subclass of blood cells is selectively tagged by incubating an aliquot of a blood sample with a first tagging agent. A second subclass of blood cells is selectively tagged by incubating the aliquot with a second tagging agent. The aliquot is then passed, without lysing of any subclasses of blood cells which are not of interest, substantially a cell at a time through an area of optical stimulation for the tagging agents. Light emitted by the tagging agents is detected, the detection being limited by gating to a threshold value related to a predetermined intensity of light by one of the tagging agents. Cells of the subclass are differentiated based on occurrence of emitted light from the tagging agents.

Method for analysis of subpopulations of blood cells

A method for calibrating an instrument for using that instrument to obtain at least one light-related signal from particles under analysis comprises directing an incident beam of light at calibration particles having one or more known characteristics related to the particles expected to be analyzed. Both a light signal and a noise signal from the calibration particles are detected. A measurement is made of the ratio of the detected light signal to the detected noise signal, and that measurement is reported. The measured ratio is then compared to a predetermined ratio which represents a threshold for minimum instrument performance. This method further includes adjusting, if the predetermined ratio has not been attained, the operation of the instrument, while the calibration particles are within the incident beam of light, until the measured ratio reaches the predetermined ratio whereby the instrument is calibrated for subsequently obtaining the light signal from particles to be analyzed. A kit of reagents for use in calibrating an instrument for performing two-color analysis of particles and particle standards for calibrating fluorescence analysis instruments are also part of the instant invention.

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Cupertino, CA, United States of America

Diether J Recktenwald