Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
1998-02-26
2001-08-28
Snay, Jeffrey (Department: 1744)
Chemistry: analytical and immunological testing
Biological cellular material tested
C436S164000, C356S073000
Reexamination Certificate
active
06281018
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates in general to fluid analysis systems, such as flow cytometers, and is particularly directed to a process for analyzing a fluid, such as one containing human blood cells, for the presence of prescribed constituents, such as preselected types of blood cells, and then sorting successively generated fluid droplets to a selected one of a plurality of collection stores based upon the constituency of a selected plurality of contiguous droplet precursor regions.
BACKGROUND OF THE INVENTION
Flow cytometers are commonly employed in the medical industry to analyze particles in a patient's body fluid (e.g., blood cells) as an adjunct to the diagnosis and treatment of disease. As a non-limiting example, in the course of chemotherapy treatment, such instruments may be used to sort and collect healthy blood cells (stencils) from a quantity of blood that has been removed from a patient's bone marrow prior to chemotherapy. Once a chemotherapy treatment session is completed, a collected quantity of these cells is then reinjected back into the patient, to facilitate migration and healthy blood cell reproduction.
For this purpose, as diagrammatically illustrated in
FIG. 1
, particles
10
to be analyzed, such as cells of a centrifuged blood sample stored in a container
12
, are analyzed by injecting the particles into a (pressurized) continuous or uninterrupted carrier fluid (e.g., saline)
14
, and directing the carrier fluid along a channel
15
that passes through the output beam
16
emitted by an optical illumination subsystem, such as one or more lasers
18
. Located optically in the path of the laser output beam
16
after its being intercepted by the carrier fluid passing therethrough are one or more photodetectors of a photodetector subsystem
21
.
This photodetector subsystem
71
is positioned to receive light as modulated by the contents of (particles/cells within) the fluid in the channel
15
, including that reflected off a cell, the blocking of light by a cell, and a light emission from a fluorescent dye antibody attached to a cell. In order to avoid confusion as to which photodetector output signal is representative of which illuminated cell, the fluid flow channel through the cytometer is configured and sized to pass the particles or cells only one cell at the time through an intersection location
19
with the laser's output beam
16
. As a consequence, as the output signals from the photodetector of interest is irradiated by the laser beam to the time when a droplet containing the cell of interest eventually separates from the carrier fluid stream.
The individual droplets
23
produced by the droplet generator
25
do not form immediately at the droplet generator's exit port
27
, but rather break off naturally and in random fashion at location
29
downstream of the exit port
27
. The point in space
29
downstream of the exit port
27
at which the droplets individually form may be adjusted by varying the parameters of the drive signal to the piezoelectric transducer of the droplet generator
25
, and may be defined so as to cause the droplets
23
to become synchronized with the frequency of the piezo vibration of the droplet generator
25
. As a non-limiting example, the acoustic drive frequency applied to the droplet generator
25
may be on the order for from four to one hundred Khz, at a fluid pressure on the order of from three to seventy psi.
Once the droplets
23
have been individually formed in a spaced apart sequence, they may be controllably sorted by means of droplet sorter
30
into a collection container
41
, or allowed to pass unsorted along a main travel path
24
into an aborted or discarded waste container
43
. The droplet sorter's electrostatic charging collar
31
may comprise a metallic ring surrounding that point in the droplet stream
22
where the individual droplets
23
separate from the fluid stream, and is typically several droplets in length. It is positioned vertically downstream of the exit port
27
of the droplet generator
25
, and upstream of an subsystem
21
are modulated by the particles in the carrier fluid, each modulation signal can be associated with a respective cell in the fluid carrier stream.
If the output of the photodetector subsystem
21
satisfies prescribed ‘sort’ criteria associated with one or more parameters of a desired cell, then subsequent to a ‘sort delay’, the photodetector output signal may be used to control the sorting of a droplet of the carrier fluid containing that cell (by means of a downstream droplet sorter
30
), once that droplet is formed by into a stream of droplets
23
by means of an acoustically (e.g., piezoelectric) driven droplet generator
25
. For this purpose, the photodetector output is typically digitized and then analyzed by a cell type mapping or identification algorithm executed by an associated supervisory control processor. Based upon this analysis, the control processor instructs the droplet sorter to sort or abort the droplet.
The sort delay is the period of time that elapses between the instant in time at which the photodetector generates an output signal for that cell as it is illuminated by the laser beam at intersection location
19
and the time at which that portion of the fluid stream containing that cell breaks off into a droplet at some downstream location
29
. The sort delay may be defined means of by a delay timer, such as a counter driven by a high speed sort control clock, so that the output of the counter indicates the time difference between the time that the portion of region of the carrier fluid containing the cell associated set of electrostatic (opposite polarity, high voltage) deflection plates
33
and
35
between which the stream of charged droplets
23
pass as they travel downwardly towards the collection and waste containers.
Under the control of a cell analysis and sorting routine executed by the system workstation
50
, a prescribed charging voltage is selectively applied via deflection control circuitry
52
to the charging collar
31
at a time determined by the sort delay, thereby charging that segment of the cell-containing fluid stream, so that any droplet breaking off from the stream at that point and containing the cell of interest will carry the charge induced by the collar. Then, as an individual charged droplet carrying this charge (one of which is shown at
23
C) passes between the two opposite polarity high voltage deflection plates
33
and
35
, it is attracted to the plate with the opposite charge, while being simultaneously repelled by the plate with the same or like charge. For a droplet containing a cell to be sorted, this electrostatic steering action directs the charged droplet
23
C along a path
26
to one side of the travel path
24
of the main stream, and into the collection container
41
placed on the side of the path of the main stream of droplets.
The location of the point
29
where an individual droplet
23
forms or breaks off from the continuous fluid stream
22
is critical to accurate sorting of the droplets, since only a droplet that breaks off from the stream at the time of the applied sort charge will be deflected by the deflection plates, and subsequently collected in the target sorting container. As described above, for any given cell
10
within the fluid stream
22
, there is a ‘sort’ delay between the time at which the photodetector subsystem
21
generates an output signal for that cell and the time at which a droplet
23
containing that cell breaks off from the fluid stream. As described previously, during this sort delay, the output signal from the photodetector is digitized and then analyzed-processed by a droplet sort routine executed by the system's control processor (controlled by or installed within workstation
50
) to determine whether the droplet containing the cell of interest is to be sorted into the collection container
41
or aborted into the waste container
43
.
Since sort delay is affected, inter alia, by the
Kirouac William E.
Starling J. David
Statler Eric
Alter Mitchell E.
Coulter International Corp.
Snay Jeffrey
Wands Charles E.
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