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Flow Cytometry with the Automated Multiparameter Analyzer for Cells, AMAC

Publications in Reverse Chronological Order.

AMAC Patents that can be downloaded.

Red Blood Cell Shape

R. S. Frank, J. L. Wyatt, W. Gong, C. M. Rodriguez, and R. C. Leif. Apparatus and Method for Determination of Individual Red Blood Cell Shape, US 5,798,827 (1998).

Abstract

The present invention provides an apparatus and method to determine individual red blood cell shape within a flow cytometric aperture. Cell shape is determined by measuring the asymmetry of a light scatter pattern produced by laser illumination of the cell within the aperture by light scatter detectors which collect light from specific angular regions of light scatter, and analyzing the asymmetry of the light scatter patters to determine the individual red blood cell shape. Embodiments of the invention further provide for the determination of red blood cell shape by measuring the asymmetry of a light scatter pattern and measuring electrical resistance of the red blood cell.

RBC Lyse

R. C. Leif, S. Ledis, and R. Fienberg, Reagent system and method for identification, enumeration and examination of classes and subclasses of blood leukocytes, US 5,188,935 (1993).

Abstract

Specified classes and subclasses of leukocyte blood cells are identified by immunohematology procedures, based on utilization of antigenic determinants on the cell surface, their reactivity with antibodies which fluoresce under known circumstances, and identified by utilization of principles of flow cytometry or morphology. This invention particularly concerns improvements in the lysing and fixing method used prior to detection and identifying of the cells. In this method, a whole blood sample first is incubated with a reagent including antibodies to the cell subclass to be identified, the antibodies having directly or indirectly made fluorescently responsive to a particular light (e.g. argon ion laser). The red blood cells then are lysed with a reagent containing saponin. Next follows a leukocyte fixing treatment, preferably using a cross-linking dialdehyde composition, such as glyoxal or glutaraldehyde. Details are given as to time, temperature, concentration, and the use of additives and stains, as well as the use of particular monoclonal antibodies in the preferred procedure.

Concave Reflecting Grating

R. C. Leif, Monitoring of a detection zone utilizing zero order radiation from a concave reflecting grating, US 4,351,611 (1982).

Abstract

Abstract Disclosed is an optical flow system wherein individual particles, which are sequentially suspended in a fluid flow, are irradiated at a detection zone with an excitation source to generate radiation signals, which in turn are dispersed by a concave reflection grating for subsequent detection and measurement. The radiation signals which are not dispersed from the grating but are reflected from the grating, are further reflected by a conjugate mirror to form a projected image of the detection zone suitable for viewing with a screen or eyepiece.

Multiparameter Transducer

R. C. Leif, Orifice inside optical element, US 4,348,107 (1982).

Abstract

Disclosed is an electro-optical transducer for simultaneously making optical measurements and electrical volume measurements on particles suspended in a flow stream passing through an orifice positioned inside an optically clear spherical element.

Cell Volume Transucer

R. C. Leif, Use of fluid retarding ion conducting material, US 4,258,316 (1981).

Abstract

An improved particle sensing transducer apparatus and method for studying the physical properties of particles suspended in an electrolyte solution. The transducer apparatus includes a first chamber, at least a portion of which contains a quantity of the electrolyte solution with a first electrode disposed therein. A second chamber is provided, at least a portion of which contains a quantity of the electrolyte solution with a second electrode disposed therein. The transducer further includes an orifice for establishing a constricted electrical path by providing a passageway for a sample flow of electrolyte solution containing the particles between the two chambers. The improvement comprises a fluid retarding, ion conducting material, such as a gel, frit or membrane, interposed between the sample flow and at least one of the electrodes so as to pass an ionic current while retarding the electrolyte flow from the electrode.

AMAC Papers that can be downloaded.

Spherical Multiparameter Transducer

R. C. Leif, M. L. Cayer, W. Dailey, T. Stribling, and K. Gordon, The use of a Spherical Multiparameter Transducer for Flow Cytometry, Cytometry 20, pp 185-190 (1995).

Abstract

Fused silicon dioxide, multiparameter flow transducers with 50 mm internal square cross section and circa 60 mm length can simultaneously measure DC and RF impedance, as well as fluorescence and multiple angle light scattering. One of these spherical "Automated Multiparameter Analyzer for Cells" transducers was mounted in an EPICSTM CVA flow cell housing and installed on a research prototype equipped with an argon ion laser. The signal produced by the spherical transducer with EPICSTM DNA-Check beads was 1.73 times greater than that produced with the standard cylindrical flow cell. Similarly, with EPICSTM Immuno- Brite beads the average ratio was 1.96. The Coulter impedance and light scattering measurements were similar to those produced with the conventional cylindrical outside flow cell, although the internal cross section of the sphere was square and that of the cylinder was circular. The theoretical arguments of Leif and Wells have been demonstrated to be correct. At present, monolithic, spherical fused silica transducers are the optimum design for combined electro-optical, multiparameter flow cytometry analyzers.

Two-dimensional impedance studies

R. C. Leif, S. Schwartz, C. M. Rodriguez, L. Pell-Fernandez, M. Groves, S. B. Leif, M. Cayer, H. Crews; Two Dimensional Impedance Studies of BSA Buoyant Density Separated Human Erythrocytes, Cytometry 6 pp. 13-21 (1985).

Abstract

Combined DC (Coulter Volume) and radio frequency impedance studies were performed on human erythrocytes which had been separated by buoyant density in linear, neutral, isotonic bovine serum albumin gradients. The individual buoyant density fractions showed no reproducible shift in volume with buoyant density but did show a shift with opacity, radio frequency impedance divided by dc impedance. This new electronic parameter of opacity can be related to cell age, since both it and cell age are directly related to buoyant density. This increase in opacity with buoyant density is correlated with a change in shape.

Instrumentation and Fluorochromes

R. C. Leif, R. A. Thomas, T. A. Yopp, B. D. Watson, V. R. Guarino D. H. K. Hindman, N. Lefkove and L. M. Vallarino; Development of Instrumentation and Fluorochromes for Automated Multiparameter Analysis of Cells, Clin. Chem. 23, 1492-1498 (1977).

Abstract

We have developed and interfaced to a computer an automated instrument (the AMAC III) which is designed to observe simultaneously several physical parameters of cells. Typical parameters include electronic cell volume (Coulter effect), RF amplitude (opacity), multiwavelength fluorescence of cytological stains, and cell light-scattering. The use of a new ultraviolet laser combined with a holographic grating spectrograph promises to increase the number of fluorescing species that can be detected simultaneously. This number can be further increased by use of special rare-earth-based fluorochromes, that emit well-defined, spectrally distinct peaks.

Electronic cell-volume analysis

R. C. Leif and R. A. Thomas; Electronic Cell Volume Analysis Utilizing the AMAC I Transducer, Clin. Chem. 19, pp. 853-870 (1973).

Abstract

We have developed and interfaced to a computer an automated instrument (the AMAC III) which is designed to observe simultaneously several physical parameters of cells. Typical parameters include electronic cell volume (Coulter effect), RF amplitude (opacity), multiwavelength fluorescence of cytological stains, and cell light-scattering. The use of a new ultraviolet laser combined with a holographic grating spectrograph promises to increase the number of fluorescing species that can be detected simultaneously. This number can be further increased by use of special rare-earth-based fluorochromes, that emit well-defined, spectrally distinct peaks.

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