Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
1999-05-13
2003-07-15
Warden, Jill (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C422S068100, C422S073000, C422S105000, C422S105000, C436S180000, C436S043000, C436S171000, C702S019000, C702S021000, C702S022000, C702S025000, C702S027000, C702S030000, C702S045000, C702S049000, C356S072000, C356S073000
Reexamination Certificate
active
06592822
ABSTRACT:
FIELD OF THE INVENTION
The present invention pertains generally to a diagnostic system and/or method, and more particularly to a substantially simultaneous and multiplexed, multi-analyte diagnostic system and/or method for performing assays using a flow analyzer.
BACKGROUND OF THE INVENTION
Flow cytometry utilizes an optical technique that analyzes particles in a fluid mixture based on the particles' optical characteristics using a flow cytometer. Background information on flow cytometry is, for example, found in Shapiro,
Practical Flow Cytometry
, Third Ed. (Alan R. Liss, Inc. 1995), incorporated herein by reference.
Conventional flow cytometers have been commercially available since the early 1970s and presently cost, for example, more than $120,000. They can be behemoths in size, occupying upwards of 13 cubic feet and weighing well over 200 pounds.
In conventional flow cytometers, as shown in
FIGS. 1 and 2
, sample fluid containing sample cells or microspheres having reactants on their surfaces is introduced from a sample tube into the center of a stream of sheath fluid. The sample fluid stream is injected into, at, or near, the center of the flow cell or cuvette. This process, known as hydrodynamic focusing, allows the cells to be delivered reproducibly to the center of the measuring point. Typically, the cells or microspheres are in suspension in the flow cell.
A continuous wave laser
1900
focuses a laser beam on them as they pass through the laser beam by a flow of a stream of the suspension. Lasers in conventional flow cytometers often require shaping a round beam into an elliptical beam to be focused on the flow cell. As shown in
FIG. 2
, this elliptical beam is often formed from the round beam using a beam shaping prismatic expander
1960
located between the laser and the flow cell.
When an object of interest
1905
in the flow stream is struck by the laser beam, certain signals are picked up by detectors. These signals include forward light scatter intensity and side light scatter intensity. In the flow cytometers, as shown in
FIGS. 1 and 2
, light scatter detectors
1930
,
1932
are located opposite the laser (relative to the cell) to measure forward light scatter intensity, and to one side of the laser, aligned with the fluid-flow/laser beam intersection to measure side scatter light intensity.
In front of the forward light scatter detector
1930
can be an opaque bar
1920
, called a beam stop, that blocks incident light from the laser. Thus, the beam stop ensures that as little of the beam as possible will interfere with the measurement by the forward light scatter detector of the relatively small amount of light which has been scattered, by the flow cell, at small angles to the beam. Forward light scatter intensity provides information concerning the size of individual cells, whereas side light scatter intensity provides information regarding the relative size and refractive property of individual cells.
Known flow cytometers, such as disclosed in U.S. Pat. No. 4,284,412 to HANSEN et al., incorporated herein by reference, have been used, for example, to automatically identify subclasses of blood cells. The identification was based on antigenic determinants on the cell surface which react to antibodies which fluoresce. The sample is illuminated by a focused coherent light and forward light scatter, right angle light scatter, and fluorescence are detected and used to identify the cells.
As described in U.S. Pat. No. 5,747,349 to VAN DEN ENGH et al., incorporated herein by reference, some flow cytometers use fluorescent microspheres, which are beads impregnated with a fluorescent dye. Surfaces of the microspheres are coated with a tag that is attracted to a receptor on a cell, an antigen, an antibody, or the like in the sample fluid. So, the microspheres, having fluorescent dyes, bind specifically to cellular constituents. Often two or more dyes are used simultaneously, each dye being responsible for detecting a specific condition.
Typically, the dye is excited by the laser beam from a continuous wave laser
1900
, and then emits light at a longer wavelength. As shown in
FIG. 1
, dichroic filters
1940
split this emitted light and direct it through optical detectors
1950
,
1952
,
1954
that can be arranged relative to the laser. The optical detectors
1950
,
1952
,
1954
measure the intensity of the wavelength passed through, respective filter. The fluorescence intensity is a function of the cells' absorption of fluorescent dye.
FIG. 2
depicts a prior art flow cytometer which uses beam splitters
1942
,
1944
,
1946
to direct light from the flow cell
1910
to photo-multiplier and filter sets
1956
,
1958
,
1959
and to side light scatter detector
1932
. This flow cytometer employs a mirror
1970
to reflect forward light scatter to forward light scatter detector
1930
.
However, I have determined that the properties of the fluorescent dyes themselves limit this flow cytometric technique to about three different wavelengths. The difference in energy, and hence wavelength, between an excitation photon and emission photon is known as Stokes shift. Generally, the larger the Stokes shift from the excitation wavelength, the broader and weaker the emission spectra
At any given excitation wavelength, I have determined that there are often only a limited number of dyes that emit a spectrum of wavelengths narrow enough and sufficiently separated enough that they are individually measurable simultaneously. Of these, there are fewer dyes still that exhibit good quantum efficiency, for example, between 5and 40%. Other values for quantum efficiency are also acceptable. For example, values of 75 to 80% are acceptable. Consequently, researchers in flow cytometry and other fields have been limited to roughly three fluorescent labels, namely, for green, yellow-orange, and red light.
The limitation on the number of fluorescent labels necessarily crimps the amount of analysis that can be done on any one sample. Therefore, for meaningful analysis, a larger quantity of sample is required and more runs of the sample through the flow cytometer must be performed. This necessarily increases the time needed to analyze the sample. However, time is often not available in an emergency room environment, for example, where a small blood sample, must be screened simultaneously for many diagnostic indicators, including therapeutic and abused drugs, hormones, markers of heart attack and inflammation, and markers of hepatic and renal function. In addition, for efficiency reasons, it is desirable to minimize the testing time to increase the number of tests that can be performed over a predetermined time interval.
One way to overcome the limitation on the number of fluorescent labels, I have determined, is to use two lasers of different frequencies, each focused on a different spot along the flow stream. Such a configuration is called a multi-station flow cytometer. As a particle passes a first laser, up to three fluorescence measurements are taken. Then, as the particle passes the second laser, up to three more measurements are taken using a time-gated amplifier at a predetermined time interval after signals have been detected at the upstream observation point.
FIG. 3
illustrates this method.
It should be noted that the upper pair of particles A, B show the lower pair of particles A, B at a later time as the particles progress upward through the flow cell; the particles themselves are the same. In this case, laser #
1
strikes particle A. A detector for Laser #
2
must wait for a particle to pass through the beam of Laser #
2
.
Despite this dual laser approach, I have determined that it is often impossible to know for certain whether the measurements are made on the same particle. Because the measurement events at the sets of detectors are separated temporally and spatially, I have discovered that, besides laser emission timing problems, even the slightest flow turbulence can mix particles in suspension, thereby increasing the likelihood that subsequent m
Gordon Brian R
Luminex Corporation
Seabold Robert R.
Villacorta Gilberto M.
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