Optics: measuring and testing – By particle light scattering – With photocell detection
Reexamination Certificate
2001-10-02
2003-09-09
Pham, Hoa Q. (Department: 2877)
Optics: measuring and testing
By particle light scattering
With photocell detection
C356S343000, C356S073000, C436S063000
Reexamination Certificate
active
06618143
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to particle discrimination by light scattering, and more particularly to a flow cytometer and method therefore that discriminates particles employing a high numerical aperture. Numerical aperture is defined as the refractive index of the medium through which light is collected multiplied by the sine value of one-half of the angle of light collection.
BACKGROUND OF THE INVENTION
The discrimination of particles is useful in numerous clinical assays including ascertaining the types and numerical quantity of cells in blood, ascertaining invasive particles in a fluid sample, such as bacteria and virus, and quantifying the density and volume of cells in a fluid sample.
One method of the above is disclosed in U.S. Pat. No. 5,017,497 issued to de Grooth et al. Referring to
FIG. 1
, the '497 Patent discloses a flow cell
2
through which cells from, for example, blood or the like, flow substantially one by one therethrough. A laser input
4
emits a polarized beam of laser light that is oriented substantially orthogonally to the flow of blood cell through flow cell
2
such that the polarized laser light impinges upon the blood cells as they pass through flow cell
2
. By “polarized” it is meant that the plane of the electric field oscillation of the laser light is uniform. An optical lens
6
has an numerical aperture of 0.6 which limits the cone of scattered light from the blood cells that can be collected to 72° or less, and practically to 50° as disclosed in the '497 Patent. The central axis of the cone of lens
6
is 90° to both the path of the polarized laser light and the flow of blood cells through flow cell
2
. The scattered light emanating from lens
6
is collimated in a manner known in the art. The scattered light now has a mixed polarization that is characteristic of the cell type. The light next passes through a beam splitter
8
that divides the light into two separate beams. A first light beam, substantially concentric with the light beam that originally emanated from lens
6
, passes through first polarization analyzer
10
. Polarization analyzer
10
is configured to pass therethrough only polarized light having a vector the same as the original laser light. The second beam emanating from beam splitter
8
is oriented substantially perpendicular to the orientation of the first beam emanating from beam splitter
8
. This second beam enters second polarization analyzer
12
. Second polarization analyzer
12
is configured to pass therethrough only light having a polarization vector substantially orthogonal to the polarization vector of the other beam from beam splitter
8
that passed through first polarization analyzer
10
. The beams that pass through first polarization analyzer
10
and second polarization
12
enter polarized detector
14
and depolarized light detector
16
, respectively. The ratio of the outputs of polarized light detector
14
and depolarized light detector
16
, based on intensity, provide the depolarization ratio.
As shown in
FIG. 4
eosinophils, a subset of leukocytes (white blood cells), depolarize the right angle of scattered light quantified by the above configuration to a greater degree than other leukocytes.
FIG. 4
is a graphical representation having the output of polarized light detector
14
as one axis and the output of depolarized light detector
16
as the axis. While the above invention does provide some useful data regarding leukocytes, and more specifically eosinophils, as shown in
FIGS. 6B
,
7
B,
8
B and
9
B, the cluster points within the eosinophil cluster (the cluster points above the angled threshold line on the graphical representation having “DEPOL” as one axis and “ORTHAGONAL” as the other axis) are quite condensed. The dense nature of the points within the eosinophil cluster results in difficulty for the computer software programs that ascertain and identify clusters to accurately identify eosinophil clusters. Additionally, this prior art configuration requires expensive optical devices such as photo multiplier tubes, and lens
6
, first polarization amplifier
10
and second polarization amplifier
12
.
The prior art as indicated in the '497 Patent is unable to distinguish eosinophils without utilizing polarized and depolarized light methods, because the cone of light collected is 72° or less, based on the numerical aperture of the light collection lens, and more practically 50°, based on the number of optical elements that are used. These optical elements, such as beam splitter
8
, polarization analyzers
10
and
12
, and light detectors
14
and
16
, contribute to reducing the effective light collection of the system is substantially less that 72°, and to more practically, 50°.
Copending U.S. patent application Ser. No. 09/507,515 discloses a device and method for distinguishing eosinophils in a sample of blood cells. The device uses a right angle scatter light detector that is effective to collect a cone of unfiltered right angle scattered light of at least 100° and convert the collected right angle scattered light into a right angle scattered light signal. This signal is processed by the device to distinguish eosinophils from other leukocytes in the sample on the basis of the right angle scattered light signal.
While the device described in the '515 application is capable of detecting eosinophils using unfiltered right angle scattered light, its properties create problems for the economical production of the device. Mechanically, providing a solid mounting scheme to keep right angle scatter detector
22
in place at a very small distance to flow cell
18
is difficult to design, and more difficult to manufacture. Also, minimizing the distance from right angle detector
22
to pre-amplifier
26
is essential to eliminate electrical noise that would otherwise be picked up by the leads of the right angle photo detector
22
.
A need thus exists for a flow cytometer apparatus and related method in which the cell cluster points are less dense for ease of characterization of the different cell clusters. A need also exists for the above apparatus and method which has fewer and less expensive components, and which is easy and economical to manufacture.
SUMMARY OF THE INVENTION
The lens-less light collection 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. Unlike the prior art, the laser light emitted by the laser input need not be polarized for analysis of the cells according to the present invention. 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 (“right angle scatter light”). 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°. This scatter is termed “forward scatter light” and is collected on two distinct photo detectors. The first detector represents ‘forward scatter low’ (FSL), i.e., forward scatter light which has an angle of from about 1° to about 3° relative to the laser beam input. A second detector represents ‘forward scatter high’ (FSH), i.e., forward scatter light which has an angle of from about 9° to about 12° relative to the laser beam input A third photo detector, axial with the impinging laser light, is placed in between these two forward scatter detectors. 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. The right angle scatter light detector is preferably located about 2 millimeters from the blood cells in the flow cell. An important aspect of the present inventi
Flynn, Jr. Harold C.
Hansen W. Peter
Roche John W.
Carter DeLuca Farrell & Schmidt LLP
Idexx Laboratories Inc.
Pham Hoa Q.
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