Note: Descriptions are shown in the official language in which they were submitted.
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TITLE: METHOD AND APPARATUS FOR SORTING AND SEPARATING
PARTICLES FROM A FLUID SUSPENSION
FIELD OF THE INVENTION
The present invention relates to cytological
specimen testing and more particularly to a method and
apparatus for sorting and separating biological cells or
material using the motive force of gas bubbles.
BACKGROUND OF THE INVENTION
In the fields of medicine and biology, the
detection of rare or unusual cells within cytological
specimens is a common activity. Two techniques are
commonly utilized for cell detection. The first technique
involves examination of a slide through a microscope and is
classified as a static detection method. The second
technique is considered dynamic and involves the use of a
flow cytometer.
The static detection technique of rare cellular
events forms the basis of a wide range of cancer screening
tests, including the well-known Pap test for cervical
cancer precursors. The static detection method involves
arranging the specimen on the surface of a microscope slide
and then visually examining the specimen under high
magnification through the microscope. The static detection
technique has the advantage of allowing repeated
examinations of the specimen before a final decision is
made. This improves the intrinsic accuracy of the test
when performed by a skilled and trained technician.
However, as the cells of interest, i.e. the rare or unusual
cells will be mixed in with perhaps hundreds of thousands
of unimportant cells, there is a strong possibility that
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the presence of the unimportant cells will interfere with
the accurate detection of the cells of interest.
The known dynamic detection methods fall into the
general category of flow cytometry. Flow cytometry is a
known technique for making rapid measurements on particles
or cells as they flow in a fluid stream past a sensing
point. By making individual cell measurements, rare events
(i.e. unusual cells or cell formations) can be extracted
from the background (i.e. cytological specimen) in a way
that is not possible using bulk measurement in the static
detection technique. However, in most cases the flow
cytometry technique only provides a single pass of the
fluid stream flow and as a result there is only one
opportunity to detect the cells of interest or the target
cells.
In the art, there remains a need for a system
which combines the accuracy of the static detection
technique with the rapid measurement time of the dynamic
flow cytometry technique.
BRIEF SUMMARY OF THE INVENTION
The present invention provides method and
apparatus which utilizes gas bubbles for sorting and
separating particles from a fluid suspension. The
invention is suitable for sorting and separating cells in
a fluid containing a biological specimen.
The cell sorting and separation system utilizes
a dynamic flow cytometric process to identify target cells
(i.e. cells of interest) in the cytological specimen which
is carried in a fluid stream. At an appropriate point, the
cells of interest are directed from the primary fluid
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stream into a collection chamber, a secondary sorting path
or into a secondary fluid stream. The diverted cells
provide an enriched specimen which may then be prepared in
a slide format and examined using static rare event
detection techniques.
According to another aspect of the invention, the
cells of interest or target cells in the primary fluid
stream are diverted by applying a gas bubble impulse at the
appropriate point in the flow, so that the diverted portion
of the cellular fluid stream contains a higher
concentration of the target cells. The gas bubble for
diverting the target cells is generated by a high-intensity
solid state laser.
In a first aspect, the present invention provides
a device for selecting and separating particles of interest
from a fluid suspension, the device comprises: (a) an input
port for receiving the fluid suspension; (b) a main
channel, the input port being formed at one end of the main
channel, and the main channel having an output port at the
other end, the main channel defining a flow path for the
fluid; (c) a bubble forming chamber, the bubble forming
chamber being connected to the main channel and being
coupled to an energy source for receiving energy to form a
bubble within fluid contained in the bubble forming
chamber; (d) an interrogation port, the interrogation port
being located between the input port and the bubble forming
chamber; and (e) a diversion channel, the diversion channel
being located across from the bubble forming chamber and
providing a conduit for particles displaced by the bubble
formed in the bubble forming chamber.
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In a second aspect, the present invention
provides a method for separating target particles from a
fluid suspension containing one or more of said particles,
the method comprises the steps of: (a) passing the fluid
suspension containing the target particles past an
interrogation point and determining the presence of one or
more of the target particles in the fluid suspension; (b)
applying an energy source to heat a portion of the fluid
suspension proximate said target particles determined in
step (a) to form a bubble in the fluid suspension;(c)
utilizing said formed bubble to divert the target particles
for further processing.
BRIEF DESCRIPTION OF THE DRAWINGS
Reference will now be made to the accompanying
drawings, which show by way of example, a preferred
embodiment of the present invention, and in which:
Fig. 1 is a diagrammatic view of a cell sorting
and separation apparatus according to the present
invention;
Fig. 2 is a schematic view of the main components
of the cell sorting and separation apparatus of Fig. 1;
Fig. 3 is a schematic plan view of the main
components of the cell sorting and separation apparatus of
Fig. 1;
Figs. 4(a) to 4(f) schematically depict the
operation of the cell sorting and separation apparatus
according to the present invention;
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Fig. 5 shows a flow chart from the method steps
for sorting and separating cells from a fluid stream
according to the present invention;
Fig. 6 shows in schematic form a cascaded
configuration for the cell sorting and separation apparatus
according to the present invention;
Fig. 7 shows in schematic form a serial
configuration for the cell sorting and separation apparatus
according to the present invention; and
Fig. 8 shows in schematic form a parallel
configuration for the cell sorting and separation apparatus
according to the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Reference is first made to Fig. 1 which shows a
cell sorting and separation device according to the present
invention and indicated generally by reference 10. As will
be described in more detail below, the cell sorting and
separation device 10 together with an external laser 11
forms a system for effectively and rapidly separating cells
of interest (i.e. target cells) from a cellular or
biological specimen, indicated generally by reference S,
carried in a fluid stream which is passed through the
device 10. In the preferred embodiment, the cell sorting
and separation device 10 comprises a cartridge with no
moving parts and the cell sorting and separation mechanism
is actuated by the external laser 11. Advantageously, the
cartridge for the cell sorting and separation device 10
lends itself to being disposable and can be manufactured
relatively cheaply in large quantities.
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Preferably, the laser 11 is situated exterior to
the cell sorting and separation apparatus. This
arrangement provides a non-contactive system in which the
laser 11 is isolated from possible contamination and the
cell sorting and separating device 10 lends itself to a
disposable device which makes the system suitable for
medical applications. Furthermore, as the cell sorting and
separating device 10 is externally actuated and with
minimal moving parts, the device 10 is well-suited to mass
manufacturing which further covers the per unit cost of the
disposable device. Other high energy output devices may be
suitably substituted for the laser 11.
As shown in Fig. 1 (and also in Figs. 2 and 3 in
which like references indicate like elements), the cell
sorting and separation device 10 comprises a cartridge 20.
The cartridge 20 is designed to be a disposable device with
no moving parts and also lends itself to mass manufacturing
processes such as plastic injection molding. On the
cartridge 20 is formed a main channel 21, a bubble chamber
22, a diversion channel 24 and an interrogation port 26.
The main channel 21 provides the primary flow path for a
cellular specimen S suspended in fluid. The main channel
21 has an input port 28 and an output port 30. The input
port 28 couples the main channel 21 to a supply vessel (not
shown) containing the cellular specimen fluid and receives
a portion of the cellular specimen fluid S. Once the
cellular specimen fluid S passes through the device 10, it
exists at the output port 30 which is connected to a waste
collection vessel (not shown). While the device 10 is
described in the context of a biological or cellular
specimen separating application, it will be understood that
the invention has wider applicability to other applications
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where selected particles are to be separated from a fluid
suspension.
The main channel 21 first takes the cellular
specimen fluid S past the interrogation port 26. The
interrogation port 26 provides a first actuation point in
the device 10 and involves using a sensing mechanism
(indicated generally by reference 27) to identify particles
or cells of interest (i.e. target cells) in the cellular
specimen fluid S. The interrogation port 26 utilizes
optical, electromagnetic or other sensing mechanisms to
identify the target cells in the fluid specimen S. The
sensing mechanism 27 at the interrogation port 26 may
comprise a laser system which measures forward and right-
angle light scattering in the cellular fluid specimen S.
Or, the sensing mechanism 27 may comprise a fluorescence
system utilizing an ultraviolet light source and a visible
light detector to measure absorption or scattering of the
ultraviolet light. Or, the sensing mechanism 27 may
comprise some other type of non-contactive measurement that
can differentiate between or among the particles in the
fluid S flowing in the main channel 21. The sensing
mechanism 27 at the interrogation port 26 produces an
output when the cells of interest are detected at the
interrogation port 26. The interrogation output is
utilized for the second actuation point in the device 10.
The second actuation point in the device 10
occurs at the bubble chamber 22. The interrogation output
is registered by a control system 12 and used to activate
the laser 11 in a timed sequence. When the target cells or
particles have moved to the bubble chamber 22, the control
system 12 activates the external laser 11 which directs a
high energy light beam into the bubble chamber 22. The
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control system 12 may comprise a hardware (e. g. logic)
circuit, a microcontroller or microprocessor suitably
operating under program (i.e. firmware) control, or a
computer system (e.g. personal computer) operating on a
known platform (e.g. Windows T'" or UNIX) .
The bubble chamber 22 includes a window 23 for
emitting the high energy light beam. The energy from the
light beam is absorbed by the cellular specimen fluid S in
the chamber 22 and the resultant heating in the fluid S
causes a bubble B to form. As the bubble B forms and grows
in the blind bubble chamber 22 the target particles or
cells are pushed into the diversion channel 24 and directed
into a processing chamber 27. The bubble chamber 22 may
include an optically absorptive pad 25 to facilitate the
heating (i.e. formation of the bubble B) in cellular fluid
specimen S which are not sufficiently absorptive to ensure
rapid formation of the bubble B.
The processing chamber 27 may simply comprise a
collection port. Or, the processing chamber 27 may include
a port 29 leading to a secondary cell sorting and
separation device as described below. Or, the diversion
channel 24 may comprise a short connection to a second
channel which runs parallel to the main channel 21 as
described in more detail according to another embodiment of
the invention.
Reference is next made to Figs. 4(a) to 4(f) and
the flow chart in Fig. 5, which both illustrate the
operation of the cell sorting and separation device 10
according to the present invention. The first step in Fig.
4(a) involves setting up the flow of cellular fluid
specimen S in the main channel 21 in order to carry the
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specimen past the interrogation port 26 (block 101 in Fig.
5). The second step in Fig. 4(b) involves sensing or
detecting a particle P (or cell) of interest as it is
carried past the interrogation port 26 (block 102 in Fig.
5). The presence of the particle P is registered by the
control system 12 and used to activate the external laser
11. As shown in Fig. 4(c), after a short interval of time
the target particle P is in position in the main channel 21
next to the bubble chamber 22 (block 104 in Fig. 5). The
next step in Fig. 4(d) involves activating the external
laser 11 to apply a short pulse of energy to the bubble
chamber 22 (block 106 in Fig. 5). The pulse of laser
energy locally heats the fluid S and induces the creation
of a bubble B in the fluid specimen S sitting in the bubble
chamber 22. As the bubble B evolves and grows in size, the
bubble B acts to push a portion of the fluid S in the main
channel 21 into the diversion channel 24 (block 108 in Fig.
5). Provided the formation of the bubble B is synchronized
to the passage of the target particle P past the bubble
chamber 22, the target particle P is re-directed into the
diversion channel 24. The particle P moves through the
diversion channel 24 to the processing chamber 27 where it
is stored or moved to another stage for further processing
(block 110 in Fig. 5).
Reference is next made to Fig. 6 which shows a
cascaded arrangement of the cell sorting and separation
apparatus according to another aspect of the present
invention. The cascaded arrangement is indicated generally
by reference 200 and comprises a first cell sorting and
separation device 210a and a second cell sorting and
separation device 210b. The devices 210a, 210b are as
described above for device 10 and include respective main
channels 221a, 221b, bubble chambers 222a, 222b, diversion
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channels 224a, 224b, interrogation ports 226a, 226b, input
ports 228a, 228b, and output ports 230a, 230b. An external
laser 211a and 211b is provided for each of the bubble
chambers 222a and 222b respectively. Alternatively, a
single laser 211 may be utilized for both devices 210a,
210b with an appropriate beam control mechanism.
As shown in Fig. 6, instead of a processing
chamber 227a, the diversion channel 224a for the first
device 210a is coupled to the input port 228b of the second
device 210b. The cascaded arrangement 200 allows the
selection of target cells or particles to be gradually
refined without comprising the rate of specimen processing.
The first cell sorting and separation device 210a provides
the primary cell sorting and separation operation and is
operated at a high speed because the target cells are a
very small fraction of the total cellular specimen. The
high flow rates achievable in the main channel 221a of the
primary device 210a minimize the processing time. While
the increased processing time is desirable, the selection
of target cells in the cellular suspension may not be as
accurate as required, and target cells together with other
non-specific cells may be driven into the diversion channel
224a. The second cell sorting and separation device 210b
provides a secondary sorting and separation operation to
further refine the specimen received from the primary
diversion channel 224a. Since the absolute quantity of
cellular specimen for sorting and separation is reduced
considerably, the flow rate in the main channel 221b of the
second cell sorting and separation device 210b can be
significantly reduced. This, in turn, improves the
selectivity of the bubble sorting and separation mechanism
and results in an enriched cellular specimen sample at the
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output 230b of the second cell sorting and separation
device 210b.
It will be appreciated that any number of cell
sorting and separation devices 210 may be cascaded together
as described above with reference to Fig. 6. The number of
devices 210 cascaded together will depend on the complexity
of the sorting and separation procedure to be performed.
Reference is next made to Fig. 7 which shows a
serial arrangement of the cell sorting and separation
apparatus according to another aspect of the present
invention. The serial arrangement is indicated generally
by reference 300 and comprises three cell sorting and
separation devices 310a, 310b, and 310c connected in a
serial arrangement as shown in Fig. 7. The individual
devices 310a, 310b, 310c are similar to the device 10
described above and include respective main channels 321a,
231b and 321c, bubble chambers 322a, 322b, 322c, diversion
channels 324a, 324b, 324c, interrogation ports 326a, 326b,
326c, input ports 328a, 328b, 328c and output ports 330a,
330b, 330c.
As shown in Fig. 7, the input port 328a for the
first device 310a provides the input port for the serial
arrangement 300. The output port 330a of the first device
310a is connected to the input port 328b of the second
device 310b. Similarly, the output port 330b of the second
device 310b is connected to the input port 328c of the
third device 310c. The output port 330c of the third
device 310c provides the output for the serial device 300.
The serial arrangement 300 of Fig. 7 results in
a device having a main channel 321 with multiple
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interrogation ports 326a, 326b and 326c, multiple bubble
chambers 322a, 322b and 322c, and multiple diversion
channels324a, 324b and 324c. As the cellular suspension
flows through the main channel 321, the outputs generated
from the respective interrogation points 326a, 326b, 326c
permit target cells or particles to be diverted into the
appropriate diversion channel 324a, 324b and 324c. This is
done by the control system issuing the appropriate signal
sequence to induce the bubbles in the respective bubble
chambers 322a, 322b and 322c.
Reference is next made to Fig. 8 which shows a
parallel configured cell sorting and separation device 400
according to another aspect of the invention. The parallel
cell sorting and separation device comprises a main channel
421 and a secondary channel 431. The main channel 421
includes a bubble chamber 422, a diversion channel 424, an
interrogation port 426, an input port 428 and an output
port 430. The secondary channel 431 is coupled to the main
channel 421 through the diversion channel 424.
For the parallel configured device 400, the flow
in the main channel 421 runs parallel to the flow in the
secondary channel 431. At low fluid flow velocities, e.g.
low Reynolds numbers, there will be little fluid exchange
between the main channel 421 and the secondary channel 431,
apart from diffusion. When a target cell is detected at
the interrogation port 426, the external laser is activated
to form a bubble in the bubble forming chamber 422. The
laser-induced bubble pushes the target cell out of the main
channel 421 and into the secondary channel 431. From the
secondary channel 431, the target cell is directed to
further processing steps.
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The cell sorting and separation device 10
according to the present invention has the advantage of
providing a fast reaction time since the gas bubble can be
created very rapidly and applied to the fluid stream. The
fast reaction times also translates into smaller effective
geometries for device 10 which may be manufactured as a
disposable.
In summary, the cell sorting and separation
device 10 according to the present invention embodies the
following features. The sorting and separation mechanism
is externally actuated which allows the device 10 to be
designed as a simple and inexpensive disposable cartridge.
The device 10 includes no moving parts that may foul or
otherwise contaminate the fluids. The device 10 is not
subject to wear. The device 10 can be operated at high-
speed. The device 10 is self-regenerating and re-setting.
The sorting and separation mechanism requires little space
and may be located in a range of positions in the
cartridge.
The present invention may be embodied in other
specific forms without departing from the spirit or
essential characteristics thereof. For instance, the
device is suitable for sorting and separating particles
other than just cells, from a fluid suspension. Therefore,
the presently discussed embodiments are considered to be
illustrative and not restrictive, the scope of the
invention being indicated by the appended claims rather
than the foregoing description, and all changes which come
within the meaning and range of equivalency of the claims
are therefore intended to be embraced therein.
