U.S. patent application number 12/679448 was filed with the patent office on 2010-08-05 for multi-sample particle analyzer and method for high throughput screening.
This patent application is currently assigned to INTELLICYT. Invention is credited to Terry Dunlay, Linda Trinkle.
Application Number | 20100197512 12/679448 |
Document ID | / |
Family ID | 41507644 |
Filed Date | 2010-08-05 |
United States Patent
Application |
20100197512 |
Kind Code |
A1 |
Trinkle; Linda ; et
al. |
August 5, 2010 |
Multi-Sample Particle Analyzer and Method for High Throughput
Screening
Abstract
Embodiments of the present invention provide a system and method
for analyzing a plurality of samples comprising obtaining with an
autosampler a plurality of samples from a first plate having a
plurality of sample wells wherein the autosampler has a plurality
of probes for sampling a set of samples and wherein each probe of
the plurality of probes is in communication with a separate flow
cytometer via a separate conduit. The plurality of samples
comprising particles is moved into a fluid flow stream for each
separate conduit. Adjacent ones of the plurality of samples are
separated from each other in the fluid flow stream by a separation
gas, thereby forming a gas-separated fluid flow stream. The
gas-separated fluid flow stream is independently guided to and
through each separate flow cytometer.
Inventors: |
Trinkle; Linda;
(Albuquerque, NM) ; Dunlay; Terry; (Albuquerque,
NM) |
Correspondence
Address: |
PEACOCK MYERS, P.C.
201 THIRD STREET, N.W., SUITE 1340
ALBUQUERQUE
NM
87102
US
|
Assignee: |
INTELLICYT
Albuquerque
NM
|
Family ID: |
41507644 |
Appl. No.: |
12/679448 |
Filed: |
April 24, 2009 |
PCT Filed: |
April 24, 2009 |
PCT NO: |
PCT/US09/41680 |
371 Date: |
March 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61080171 |
Jul 11, 2008 |
|
|
|
Current U.S.
Class: |
506/7 ;
506/39 |
Current CPC
Class: |
B01L 2300/0832 20130101;
G01N 35/08 20130101; B01L 3/50273 20130101; B01L 3/502761 20130101;
B01L 2300/0877 20130101; G01N 35/1074 20130101; B01L 2400/0481
20130101; G01N 35/1067 20130101; B01L 2300/0829 20130101; G01N
35/1067 20130101; B01L 2200/0684 20130101; G01N 2015/0019 20130101;
G01N 35/1095 20130101; G01N 15/14 20130101; B01L 3/502715 20130101;
G01N 15/1404 20130101; G01N 35/1074 20130101 |
Class at
Publication: |
506/7 ;
506/39 |
International
Class: |
C40B 30/00 20060101
C40B030/00; C40B 60/12 20060101 C40B060/12 |
Claims
1. A method for analyzing a plurality of samples, comprising:
obtaining with an autoampler a plurality of samples from a first
plate having a plurality of sample wells wherein the autosampler
comprises a plurality of probes for sampling the plurality of
samples and wherein each probe of the plurality of probes is in
communication with a separate flow cytometer via a separate
conduit; moving the plurality of samples comprising particles into
a fluid flow stream for each separate conduit; separating adjacent
ones of the plurality of samples from each other in the fluid flow
stream by a separation gas, thereby forming a gas-separated fluid
flow stream; independently guiding the gas-separated fluid flow
stream to and through each separate flow cytometer; operating each
separate flow cytometer to focus the gas-separated fluid flow
stream and to selectively analyze the particles in each of the
plurality of samples as the gas-separated fluid flow stream passes
through each separate flow cytometer.
2. The method of claim 1 wherein the first plate is positioned on a
platform that is moveable in the xyz direction to bring the samples
to the probes.
3. The method of claim 1 wherein the step of moving the plurality
of samples are moved by a fluid moving device located before or
after each separate flow cytometer.
4. The method of claim 1 wherein the fluid moving device is a
multihead peristaltic pump.
5. The method of claim 1 wherein the step of moving is selected
from moving with gravity, moving with suction, moving with pumping
or moving with pushing.
6. The method of claim 1 wherein the separate conduit is
tubing.
7. The method of claim 1 wherein the probes are positionable to the
samples on the first plate.
8. The method of claim 1 wherein at least one of the probes of the
plurality of probes is positionable to a sample on a second
plate.
9. The method of claim 1 wherein the step of moving the plurality
of fluid samples streams is at a rate that is independently
controlled.
10. A method for analyzing a plurality of samples, comprising:
obtaining with an autoampler a plurality of samples from a first
plate having a plurality of sample wells wherein the autosampler
comprises a plurality of probes for sampling a set of samples and
wherein each probe of the plurality of probes is in communication
with a multi-channel flow cytometer via a separate conduit attached
to each of the probes of the plurality of probes; moving the
plurality of samples comprising particles into a fluid flow stream
for each separate conduit; separating adjacent ones of the
plurality of samples from each other in the fluid flow stream by a
separation gas, thereby forming a gas-separated fluid flow stream;
independently guiding the gas-separated fluid flow stream to and
through the multi-channel flow cytometer; operating the
multi-channel flow cytometer to focus the gas-separated fluid flow
stream and to selectively analyze the particles in each of the
plurality of samples as the gas-separated fluid flow stream passes
through the multi-channel flow cytometer.
11. The method of claim 10 wherein the first plate is positioned on
a platform that is moveable in the xyz direction to bring the
samples to the probes.
12. The method of claim 10 wherein the step of moving the plurality
of samples is with a fluid moving device located before or after
the multi-channel flow cytometer.
13. The method of claim 12 wherein the fluid moving device is a
multihead peristaltic pump.
14. The method of claim 10 wherein the step of moving is selected
from moving with gravity, moving with suction, moving with pumping
or moving with pushing.
15. The method of claim 10 wherein the separate conduit is
tubing.
16. The method of claim 10 wherein the first plate is positioned on
a platform that is moveable in the xyz direction to bring the
samples to the probes.
17. The method of claim 10 wherein the plurality of probes are
positionable and move to the samples on the first plate.
18. The method of claim 10 wherein at least one of the probes of
the plurality of probes is positionable and moves to a plurality of
samples located on a second plate.
19. The method of claim 10 wherein the step of moving the plurality
of fluid samples streams is at a rate that is independently
controlled.
20. A flow cytometry apparatus comprising: an autosampler
comprising a plurality of probes with each probe suitable for
inserting a plurality of samples comprising particles from a
plurality of respective source wells into a separate fluid flow
stream; a plurality of flow cytometers in communication with the
plurality of probes of the autosampler via a separate conduit
connecting a probe of the plurality of probes with a separate flow
cytometer of the plurality of flow cytometers; and moving the
plurality of samples in each separate fluid flow streams through
each separate conduit to a selected flow cytometer of the plurality
of flow cytometers, the plurality of probes introducing aliquots of
a separation fluid between successive ones of the plurality of
samples in each of the separate fluid flow stream to configure each
of the separate fluid flow streams as a gas-separated fluid flow
stream, each of the plurality of flow cytometers focusing the
gas-separated fluid flow stream delivered by the separate conduit
and selectively analyzing the particles in each of the plurality of
samples as the gas-separated fluid flow stream passes through each
separate cytometer of the plurality of cytometers.
21. The apparatus of claim 20 wherein the plurality of probes are
positioned on an arm.
22. The apparatus of claim 20 wherein the plurality of probes are
positionable relative to the plate.
23. The apparatus of claim 21 wherein the arm is fixed relative to
the position of the plate.
24. The apparatus of claim 20 wherein the probes are independently
positionable relative to each other.
25. The apparatus of claim 20 further comprising a fluid movement
device.
26. The apparatus of claim 25 wherein the fluid movement device is
a pump.
27. The apparatus of claim 25 wherein the fluid movement device is
positioned before or after a flow cytometer.
28. The apparatus of claim 26 wherein the pump is a peristaltic
pump.
29. The apparatus of claim 20 wherein the step of moving is
selected from pumping, pushing, suctioning or moving with
gravity.
30. The apparatus of claim 29 wherein the fluid movement device
comprises a plurality of fluid movement devices each associated
with the separate conduit.
31. A flow cytometry apparatus comprising: an autosampler
comprising a plurality of probes for inserting a plurality of
samples comprising particles from a plurality of respective source
wells into a plurality of fluid flow streams; a multi-channel flow
cytometer in communication with the plurality of probes of the
autosampler via a plurality of separate conduits connected to one
each of the probes of the plurality of probes wherein each of the
separate conduits of the plurality moves a separate fluid flow
stream of the plurality of fluid flow streams; and moving the
plurality of samples in each of the separate fluid flow streams
moving in each separate conduit to the multi-channel flow
cytometer, the autosampler introducing aliquots of a separation
fluid between successive ones of the plurality of samples in the
fluid flow stream to configure the fluid flow stream as a
gas-separated fluid flow stream, the multi-channel flow cytometer
focusing the gas-separated fluid flow stream delivered by the
conduit from the autosampler and selectively analyzing the
particles in each of the plurality of samples as the gas-separated
fluid flow stream passes through the multi-channel flow
cytometer.
32. The apparatus of claim 31 wherein the plurality of probes are
positioned on an arm.
33. The apparatus of claim 31 wherein the plurality of probes are
positionable relative to the plurality of respective source
wells.
34. The apparatus of claim 32 wherein the arm is fixed relative to
the plurality of respective source wells.
35. The apparatus of claim 31 wherein the probes are independently
positionable relative to each other and the plurality of respective
source wells.
36. The apparatus of claim 31 further comprising a fluid movement
device.
37. The apparatus of claim 36 wherein the fluid movement device is
a pump.
38. The apparatus of claim 37 wherein the pump is positioned before
or after the multi-channel flow cytometer.
39. The apparatus of claim 37 is a peristaltic pump.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of the
filing of U.S. Provisional Patent Application Serial No.
61/080,171, entitled "Multi-Sample Particle Analyzer System and
Method for High-Throughput Screening", filed on Jul. 11, 2008, and
the specification and claims thereof are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
Field of the Invention (Technical Field)
[0002] The present invention relates to a particle analyzer and
sample handling for use with particle analysis apparatus and a
method for high-throughput analysis using the same.
[0003] Particle analysis using flow cytometry is used to
characterize cells and particles by making measurements on each at
rates up to thousands of events per second. The measurement
consists of simultaneous detection of the light scatter and
fluorescence associated with each event. Commonly, the fluorescence
characterizes the expression of cell surface molecules or
intracellular markers sensitive to cellular responses to drug
molecules. The technique often permits homogeneous analysis such
that cell associated fluorescence can often be measured in a
background of free fluorescent indicator. The technique often
permits individual particles to be sorted from one another.
[0004] However, a deficiency with conventional flow cytometry is
that it does not allow for the analysis of multiple samples
consisting of multiple cells or particles in a rapid manner, a fact
that has limited the uses of flow cytometry in drug discovery and
other high throughput screening applications. For example, the
industrial standard for high throughput drug discovery is 100,000
samples per day. Because of its low throughput, flow cytometry has
generally not been considered applicable to high throughput
screening applications in areas such as drug discovery, antibody
hybridoma screening, and systems biology.
[0005] There have been several efforts at automated sample handling
in flow cytometry. For example, sample handling systems are known
that use carousels to handle samples from standard sized tubes and
sample injection systems which handle samples from 96-384 well
microplates. These systems treat each tube or well as a single
sample. A separate data file is created for each sample. These
systems typically intake samples at a rate of approximately 1 up to
5 samples per minute and require priming the sample line with each
individual specimen before analysis. Therefore, a single data file
exists for each well sample interrogated.
[0006] Other groups have also used valves and syringes in flow
cytometry, most notably, the "flow injection" group, Lindberg et
al. at University of Washington. However, the processes described
above did not address throughput speed. A group at the University
of New Mexico has used high throughput flow cytometry and achieved
sampling rates as high as 40 samples per minute, see U.S. Pat. Nos.
6,878,556, 6,890,487, 7,368,084, the entire disclosure and contents
of which is hereby incorporated by reference. However, to our
knowledge, no one has reported the capability to process samples at
rates higher than 40 samples per minute for flow cytometry.
[0007] The presence of uncontrolled bubbles in the flow cytometer
system is one of the primary sources of corrupted experimental
data. Flow cytometers may periodically experience bubbles in the
sheath fluid or sample fluid lines. Precautions are taken to
exclude bubbles as bubbles are known to cause anomalies in the flow
within the flow cytometer system that reduce the performance of the
flow cytometer. Furthermore, bubbles passing through the
interrogation zone of the flow cytometer can cause spurious or
false event signals that corrupt the experimental data being
collected. The user can take corrective action only after the
bubbles have been detected, which often occurs after experimental
data has been corrupted and the user has been inconvenienced.
Therefore the flow cytometry art teaches against introduction of
bubbles in the flow stream.
SUMMARY OF THE INVENTION
[0008] It is an aspect of the present invention to provide a flow
cytometry apparatus that meets the needs of high throughput
screening for multiwall, for example, 96, 384, or 1536 well
microplates.
[0009] It is an aspect of the present invention to provide a method
for analyzing a plurality of samples in a single source file using
high throughput screening.
[0010] It is another aspect of the present invention to separate
with a gas a plurality of adjacent samples in a fluid flow stream
which passes through a flow cytometer for analysis.
[0011] One embodiment of the present invention provides for a
method for analyzing a plurality of samples comprising obtaining
with an autoampler a plurality of samples from a first plate having
a plurality of sample wells wherein the autosampler has a plurality
of probes for sampling a set of samples and wherein each probe of
the plurality of probes is in communication with a separate flow
cytometer via a separate conduit. The plurality of samples
comprising particles is moved into a fluid flow stream for each
separate conduit. Adjacent ones of the plurality of samples are
separated from each other in the fluid flow stream by a separation
gas, thereby forming a gas-separated fluid flow stream. The
gas-separated fluid flow stream is independently guided to and
through each separate flow cytometer.
[0012] Another embodiment of the present invention provides for a
method for analyzing a plurality of samples comprising the steps of
obtaining with an autoampler a plurality of samples from a first
plate having a plurality of sample wells wherein the autosampler
has a plurality of probes for sampling a set of samples and wherein
each probe of the plurality of probes is in communication with a
single multi-channel flow cytometer via a separate conduit attached
to each of the probes in the plurality of probes. The plurality of
samples comprising particles are moved into a fluid flow stream for
each separate conduit. Adjacent ones of the plurality of samples
are separated from each other in the fluid flow stream by a
separation gas, thereby forming a gas-separated fluid flow stream.
The gas separated fluid flow stream is independently guided to and
through the single multi-channel flow cytometer. The multi-channel
flow cytometer is operated to focus the gas-separated fluid flow
stream and to selectively analyze the particles in each of the
plurality of samples as the gas-separated fluid flow stream passes
through the multi-channel flow cytometer.
[0013] In a preferred embodiment, the step of moving may occur with
gravity, or suction, or pumping or pushing. In another preferred
embodiment, moving the plurality of samples is with a fluid moving
device located before or after the cytometer. For example the fluid
moving device is a multihead peristaltic pump. The conduit through
which the fluid flow streams move may be tubing. The step of moving
the plurality of fluid flow streams through each separate conduit
is at a rate that may be independently controlled for each
conduit.
[0014] In another preferred embodiment the first plate is
positioned on a platform that is moveable in the xyz direction to
bring the samples to the probes. Alternatively the plurality of
probes are positionable and move to the samples on the first plate.
In another embodiment a probe of the plurality of probes is
positionable and moves to a plurality of samples located on a
second plate. Further still, two or more probes may or may not
occupy the same sample well at the same time.
[0015] Yet another embodiment to the present invention provides for
a flow cytometry apparatus comprising an autosampler comprising a
plurality of probes with each probe suitable for inserting a
plurality of samples comprising particles from a plurality of
respective source wells into a separate fluid flow stream. A
plurality of flow cytometers in communication with the plurality of
probes of the autosampler via a separate conduit connecting one
probe of the plurality of probes with one flow cytometer of the
plurality of flow cytometers. Moving the plurality of samples in
each separate fluid flow streams through each separate conduit to a
selected cytometer of the plurality of flow cytometers, the
plurality of probes introducing aliquots of a separation fluid
between successive ones of the plurality of samples in each of the
separate fluid flow streams to configure each of the separate fluid
flow streams as a gas-separated fluid flow stream, each of the
plurality of flow cytometers focusing the gas-separated fluid flow
stream delivered by the separate conduit and selectively analyzing
the particles in each of the plurality of samples as the
gas-separated fluid flow stream passes through each separate
cytometer of the plurality of cytometers. In a preferred embodiment
of the present invention the plurality of probes are positioned on
a bracket. Alternatively, the plurality of probes are positionable
relative to the plurality of respective source wells and/or the
bracket is fixed relative to the plurality of respective source
wells and/or the probes are independently positionable relative to
each other and the plurality of respective source wells. In a
preferred embodiment, the apparatus further comprises a fluid
movement device which may be a pump such as a peristaltic pump. The
pump may be positioned before or after each flow cytometer. The
fluid movement device comprises a plurality of fluid movement
devices each one of the plurality of fluid movement devices may be
associated with a separate conduit. In a preferred embodiment the
step of moving is selected from pumping, pushing, suctioning or
moving with gravity.
[0016] Yet another embodiment provides for a flow cytometry
apparatus comprising an autosampler comprising a plurality of
probes for inserting a plurality of samples comprising particles
from a plurality of respective source wells into a plurality of
fluid flow streams. A multi-channel flow cytometer is in
communication with the plurality of probes of the autosampler via a
plurality of separate conduits connected to one each of the probes
of the plurality of probes wherein each of the separate conduits of
the plurality moves a separate fluid flow stream of the plurality
of fluid flow streams. The plurality of samples moving in each of
the separate fluid flow streams moving in each separate conduit
moves to the multi-channel flow cytometer with the autosampler
introducing aliquots of a separation fluid between successive ones
of the plurality of samples in the fluid flow stream to configure
the fluid flow stream as a gas-separated fluid flow stream. The
multi-channel flow cytometer focusing the gas-separated fluid flow
stream delivered by the conduit from the autosampler and
selectively analyzing the particles in each of the plurality of
samples as the gas-separated fluid flow stream passes through the
multi-channel flow cytometer.
[0017] Yet another embodiment to the present invention provides for
a flow cytometry apparatus comprising an autosampler comprising a
probe with each probe suitable for inserting a sample comprising
particles from a source well into a fluid flow stream. A flow
cytometer in communication with the probe of the autosampler via a
conduit connecting the probe with the flow cytometer. Moving the
samples in the fluid flow stream through the conduit to the
cytometer, the probe introducing aliquots of a separation fluid
between successive ones of the plurality of samples in the fluid
flow streams to configure the fluid flow stream as a gas-separated
fluid flow stream, each of the plurality of flow cytometers
focusing the gas-separated fluid flow stream delivered by the
separate conduit and selectively analyzing the particles in each of
the plurality of samples as the gas-separated fluid flow stream
passes through the flow cytometer. The apparatus further comprising
a fluid movement device located after the cytometer such that the
fluid movement device assists the autosampler with introducing
aliquots of a separation fluid between successive ones of the
plurality of samples. In an alternative embodiment the gas is
pushed into the sample via pressurization.
[0018] Additional objects and advantages of the present invention
will be apparent in the following detailed description read in
conjunction with the accompanying drawing figures.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0019] The accompanying drawings, which are incorporated into and
form a part of the specification, illustrate one or more
embodiments of the present invention and, together with the
description, serve to explain the principles of the invention. The
drawings are only for the purpose of illustrating one or more
preferred embodiments of the invention and are not to be construed
as limiting the invention. In the drawings:
[0020] FIG. 1 illustrates a particle analysis apparatus according
to one embodiment of the present invention.
[0021] FIG. 2 illustrates a particle analysis apparatus according
to another embodiment of the present invention.
[0022] FIG. 3 illustrates a particle analysis apparatus according
to another embodiment of the present invention.
[0023] FIG. 4 illustrates a particle analysis apparatus according
to another embodiment of the present invention.
[0024] FIG. 5 illustrates a particle analysis apparatus according
to another embodiment of the present invention.
[0025] FIG. 6 illustrates a particle analysis apparatus according
to another embodiment of the present invention.
[0026] FIG. 7 illustrates a particle analysis apparatus according
to another embodiment of the present invention.
[0027] FIG. 8 illustrates a particle analysis apparatus according
to another embodiment of the present invention.
[0028] FIG. 9 illustrates a particle analysis apparatus according
to another embodiment of the present invention.
[0029] FIG. 10 illustrates a particle analysis apparatus according
to another embodiment of the present invention.
[0030] FIG. 11 illustrates a particle analysis apparatus according
to another embodiment of the present invention.
[0031] FIG. 12 illustrates a particle analysis apparatus according
to another embodiment of the present invention.
[0032] FIG. 13 illustrates cytometry results of forward scatter vs.
side scatter with a system and method according to one embodiment
of the present invention.
[0033] FIG. 14 illustrates cytometry results of fluorescence vs.
time with a system and method according to one embodiment of the
present invention.
[0034] FIG. 15 illustrates a particle analysis apparatus according
to another embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0035] It is advantageous to define several terms before describing
the invention. It should be appreciated that the following
definitions are used throughout this application.
Definitions:
[0036] Where the definition of terms departs from the commonly used
meaning of the term, applicant intends to utilize the definitions
provided below, unless specifically indicated.
[0037] For the purposes of the present invention, the term
"particles" refers to any particles that may be detected using a
particle analyzer such as a cytometer where particles are focused
hydrodynamically, acoustically or with capillary action.
[0038] For the purposes of the present invention, the term "source
well" refers to any container that is intended to hold a fluid such
as a fluid sample, for example a well on a well plate, whether or
not the source well contains a sample. For the purposes of the
present invention, the term "sample source well" refers to a source
well containing a sample.
[0039] For the purposes of the present invention, the term "sample"
refers to a fluid solution or suspension containing particles to be
analyzed using a method and/or apparatus of the present invention.
The particles to be analyzed in a sample may be tagged, such as
with a fluorescent tag. The particles to be analyzed may also be
bound to a bead, a receptor, or other useful protein or
polypeptide, or may just be present as free particles, such as
particles found naturally in a cell lysate, purified particles from
a cell lysate, particles from a tissue culture, etc. The sample may
include chemicals, either organic or inorganic, used to produce a
reaction with the particles to be analyzed. When the particles to
be analyzed are biomaterials, drugs may be added to the samples to
cause a reaction or response in the biomaterial particles. The
chemicals, drugs or other additives may be added to and mixed with
the samples when the samples are in sample source wells or the
chemicals, drugs or other additives may be added to the samples in
the fluid flow stream after the samples have been intaken by the
autosampler.
[0040] For the purposes of the present invention, the term
"adjacent samples" refers to two samples in a fluid flow stream
that are separated from each other by a separation gas, such as an
air bubble. For the purposes of the present invention, the term
"immediately adjacent samples" refers to adjacent samples that are
only separated from each other by a separation gas. For the
purposes of the present invention, "buffer fluid separated adjacent
samples" refers to adjacent samples that are separated from each
other by two separation gas bubbles and a buffer fluid, with the
buffer fluid being located between the two separation gas
bubbles.
[0041] For the purposes of the present invention, the term
"separation gas" refers to any gas such as air, an inert gas, or
fluid, etc. that can be used to form a gas bubble or immiscible
fluid between adjacent samples or between a sample and a buffer
fluid. An immiscible fluid is a fluid that will not substantially
mix with and contaminate a sample.
[0042] For the purposes of the present invention, the term "buffer
fluid" refers to a fluid that is substantially free of the
particles to be detected by the apparatus and method of the present
invention.
[0043] For the purposes of the present invention, the term
"plurality" refers to two or more of anything, such as a plurality
of samples. For the purposes of the present invention, the terms
"a", "an" or "the" refers to one or more of anything, such as a
sample or the sample.
[0044] For the purposes of the present invention, the term
"homogenous" refers to a plurality of identical samples. The term
"homogenous" also refers to a plurality of samples that are
indistinguishable with respect to a particular property being
measured by an apparatus or a method of the present invention.
[0045] For the purposes of the present invention, the term
"heterogeneous" refers to a plurality of samples in a fluid flow
stream in which there are at least two different types of samples
in the fluid flow stream. One way a heterogeneous plurality of
samples in a fluid flow stream of the present invention may be
obtained is by intaking different samples from different source
wells in a well plate. The identification of sequential samples to
be analyzed in the fluid flow stream is based upon the separation
of the fluid samples by one or more bubbles of a separation gas
and/or one or more portions of a buffer fluid. Another way of
obtaining a heterogeneous plurality of samples is by intaking
different samples from identical source wells at various time
points where a reaction or a series of reactions is or had been
occurring.
[0046] For the purposes of the present invention, the term "fluid
flow stream" refers to a stream of fluid samples, separated by one
or more bubbles of a separation gas and/or one or more portions of
a buffer fluid.
[0047] For the purpose of the present invention, the term "fluid
flow path" refers to device such as a conduit, tube, channel, etc.
through which a fluid flow stream flows. A fluid flow path may be
composed of several separate devices, such as a number of connected
or joined pieces of tubing or a single piece of tubing, alone or in
combination with channels or other different devices.
[0048] For the purposes of the present invention, the term "high
speed multi-sample tube" includes any tube that may be used with a
fluid movement device such as a peristaltic pump, a syringe pump,
an injector, or gravity. The tubing for use with a peristaltic pump
for example may have compression characteristics that allow a
peristaltic pump to move samples separated by a separation gas
through the tube at a speed of at least 6 samples per minute
without causing adjacent samples to mix with each other.
DETAILED DESCRIPTION OF THE INVENTION
[0049] An embodiment of the present invention preferably uses a
separation fluid, such as air, water, bubbles, or a combination
thereof to separate samples introduced from an autosampler into a
conduit such as a tubing line that connects directly or indirectly
the autosampler and a particle analyzer such as a flow cytometer
using sheath fluid or acoustic forces or capillary action to focus
particles to an interrogation zone of a flow cell. In one
embodiment, the fluid is moved from the autosampler to the particle
analyzer. The fluid sample is pulled or pushed through a conduit by
for example by pump action, suction, syringe or by gravity. A
separation fluid such as a gas, preferably air bubbles, effectively
separates a sample into a plurality of samples or separates a first
sample from a second sample which have previously occupied
different sample source well locations on a plate. Samples
separated by air bubbles are analyzed by the particle analyzer as a
continuous fluid sample. Data from the optical signature of a
plurality of samples in the fluid sample stream is collected in a
single data file. An interrogation zone is an area within a
particle analyzer where the particles are aligned or focused. A
light source such as a laser and a light detector are connected to
the interrogation zone such that a sample flowing through the
interrogation zone can be optically analyzed through methods known
in the art of flow cytometry. The particle analyzer may or may not
contain a flow cell.
[0050] FIG. 1 illustrates a preferred multi-sample particle
analyzer apparatus 100 of one embodiment of the present invention.
Multi-sample particle analyzer apparatus 100 preferably comprises
conventional autosampler, for example a gantry robot, having
adjustable XYZ positioning arm 102 on which is positioned a
plurality of hollow probes 104a-d. As arm 102 moves back and forth
(left and right in FIG. 1) and side to side (into and out of the
plane of FIG. 1), one or more of a plurality of probes selected
from 104a-d are lowered into different individual source wells
108a-g of plate 106 to obtain a first sample. The movement of the
sample probes with regards to the sample source wells is programmed
by a user into a computer programmed with instructions for moving
the arm in the XYZ directions in space. The first sample may have
been spiked with particles of different size or tags having a
fluorescent property that will be detected upon interrogation of
the sample with a light source such as a laser. One or more of
probes 104a-d uptakes a sample from a separate well 108a-d.
[0051] Once a sample is picked up by one or more of the plurality
of probes 104a-d, the sample is moved through a tube 110a-d in
communication with the corresponding probe selected from 104a-d
such that the tube extends from autosampler XYZ arm 102 and into a
multi-channel fluid movement device such as a multihead peristaltic
pump or multiple injectors 120 and then into a particle analyzer
130, 132, 134, 136 in communication with the respective tube
110a-d. A particle analyzer 130, 132, 134, 136 may comprise one or
more of the following: a flow cell, a means for focusing the
particles to a desired location within the flow cell for the sample
to be analyzed, a laser interrogation device, an optical system
configured to receive and focus the light from a laser source, a
spectrum detector, a fiber optic transmission means, processor for
collecting data from particles, and data storage. A laser
interrogation device examines individual samples flowing through a
flow cell located therein at a laser interrogation point. In
between intaking sample material from each of source wells 108, one
or more of the plurality of probes 104a-d is allowed to intake a
fluid such as air, thereby forming a gas separation between each
adjacent sample. The continuous fluid flow stream from each of the
probes 104a-d and through the respective tube 110a-d are
communicated to a respective individual particle analyzer for
example 130, 132, 134, 136. In a preferred embodiment the fluid
movement device is positioned after the particle analyzer and
therefore the sample moves from the sample probe to the particle
analyzer through the tube.
[0052] When a fluid sample having particles therein passes through
a laser interrogation point, the particles in the fluid sample are
sensed by the particle analyzer for example 130 of FIG. 1 due to,
for example, a fluorescent tag on the particles or the light
scattering properties of the particles themselves. In contrast,
when air bubbles pass through laser interrogation point no
particles are sensed. Therefore, a graph of the data points of
fluorescence sensed versus time for a series of samples analyzed
using the particle analyzer of the present invention will form
distinct groups, each aligned with the time that a sample
containing particles passes through the laser interrogation
point.
[0053] In order to detect the presence of each of two or more
different types of samples, in a heterogeneous plurality of
samples, each of the two or more different types of samples may be
tagged with different fluorescent tags, different amounts of a
single tag or some combination of different tags and different
amount of a single tag. In such a case, the groupings of data
points will vary vertically on a fluorescence versus time graph,
depending on which type of sample is being sensed. As with the case
of sensing a single type of sample, each sensed sample will exhibit
a group of data points aligned with the time that the sample passes
through the laser interrogation point.
[0054] In one embodiment of the device, samples from selected wells
in the well plate are introduced as a single specimen to a particle
analyzer and data from all of the wells in the plate or a portion
of a plate are collected in a single data file by the particle
analyzer system. In a preferred embodiment, sample from every well
having sample herein in the plate is introduced as a single
specimen in a continuous manner. In the case of multiprobe sample
introduction, multiple analyzers may create multiple data files
that the analysis protocol will associate with the well from which
the sample was taken. Multichannel sampling from a single plate
into multiple particle analyzers, or multiplate data from multiple
analyzers are analyzed by proprietary software that correlates the
data with individual well information.
[0055] In an alternative embodiment of the present invention using
the particle analyzer apparatus of FIG. 1, some of the source wells
on the well plate of the apparatus illustrated in FIG. 1 may
contain a buffer solution to allow for the formation of buffer
fluid separated adjacent samples in a tube through which samples
pass. When this is the case, after the first sample is taken up by
the probe from a first well, the probe intakes air, then buffer
solution, located in second well, then the probe uptakes air again,
and then the probe uptakes a second sample, located in a third
well. This sequence may then be repeated for additional samples
located in subsequent wells, which the probe subsequently
intakes.
[0056] A graph of the data points of fluorescence detected versus
time for a series of samples analyzed using one embodiment of the
particle analyzer of the present invention will form distinct
groups, each aligned with the time that a sample containing
particles passes through the laser interrogation point (see for
example FIG. 14). In order to detect the presence of two or more
different types of samples, each of the two or more different types
of samples may be tagged with different fluorescence tags or
different amounts of a single tag as compared to the particle in
the sample. In such a case, the groupings of data points will vary
vertically on a fluorescence versus time graph, depending on which
type of sample is being sensed. As with the case of sensing a
single type of sample, each sensed sample will exhibit a group of
data points aligned with the time that the sample passes through
the laser interrogation point.
[0057] Alternatively, buffer fluid separated adjacent samples may
be formed by providing a reservoir of buffer fluid in the
autosampler or attached to the autosampler to inject buffer fluid
into the tube for the fluid flow stream. In this case, after each
sample is taken up by the probe, the probe intakes air, then buffer
fluid is injected into the tube for the fluid flow stream, then the
probe intakes air again, and then the probe intakes a second
sample. This sequence may then be repeated for subsequent samples
to be separated by a buffer fluid.
[0058] One embodiment of the present invention is compatible with
standard commercial multi-well plates for use with autosamplers
from 96 well plates to 1536 well plates or greater. The source
wells of one embodiment of the present invention may be all filled
with samples and/or buffer fluids, or some may be left empty or
some combination thereof. When there is a plurality of different
types of samples in the source wells of a well plate, the sample
types may be arranged in the order in which they are taken up by
the probe, or the sample types may be arranged in any other
convenient arrangement. For example, all of the source wells in a
first row of source wells may contain one sample type and all of
the source wells of a second row may contain a second sample type.
Or individual wells may contain multiple specimens that are
differentiated by size of particles within the sample or
fluorescent probes within the sample associated with particles
therein.
[0059] The source wells may be of any conventional shape used for
source wells in a well plate for an autosampler. Preferably, when
small amounts of sample are used in each source well, the source
wells are conical in shape, to allow even the smallest amounts of
sample to be withdrawn by the probe or to allow the particles to
concentrate in the bottom of the well. The use of a well plate with
conical source wells reduces the problems associated with the
settling of particles to the bottom of the well prior to being
intaken by the probe. A device which resuspends the particles
before and during sampling such as an orbital shaker can be used to
reduce issues associated with settling of particles and is being
used in the current embodiment of the invention. FIG. 2 illustrates
an alternative embodiment of the multi-sample particle analyzer
apparatus 200 of the present invention. Multi-sample particle
analyzer apparatus 200 preferably comprises conventional XYZ stage
202 on which is positioned a well plate 206. The plurality of
probes 204a-d are stationary as stage 202 moves back and forth
(left and right in FIG. 2) and side to side (into and out of the
plane of FIG. 2), the stage 202 raises and lowers the well plate
206 to a plurality of probes 204a-d so that individual samples from
individual source wells 208a-g are obtained from well plate 206.
Alternatively, the probes may be positionable and move to the
sample well. Further still, the probes and the well plate may move
with respect to each other in a coordinated fashion.
[0060] Once a sample is picked up by one or more of the plurality
of probes 204a-d, the sample is moved through a tube 210a-d in
communication with the corresponding probe selected from 204a-d
such that the tube extends from the respective probe 204a-d into a
multi-channel fluid movement device 220 and then into the particle
analyzers in communication with the respective tube 210a-d.
However, the fluid movement device 220 is not required to be
located between the sample probe and the particle analyzer as the
fluid movement device may be located after the particle analyzer or
may be absent altogether in the case of gravity moving the samples.
A particle analyzer 230, 232, 234, 236 comprises a flow cell and a
laser interrogation device. A laser interrogation device examines
individual samples flowing through a flow cell located therein at a
laser interrogation point. In between taking up sample material
from each of source wells 208a-d for example, one or more of the
plurality of probes 204a-d is allowed to intake (sometimes referred
to herein as uptake) a fluid such as air prior to moving to a
subsequent well, thereby forming a gas separation between each
adjacent sample. The continuous fluid flow stream from each of the
probes 204a-d and through the respective tube 210a-d are
communicated to a respective individual particle analyzer 230, 232,
234, 236. Multichannel fluid movement device may be for example a
multihead peristaltic pump or a multichannel push-pull syringe
system.
[0061] FIG. 3 illustrates an alternative embodiment of the
multi-sample particle analyzer apparatus 300 of the present
invention. Multi-sample particle analyzer apparatus 300 preferably
comprises conventional autosampler having adjustable XYZ
positioning arm 302 on which is positioned a plurality of hollow
probes 304a-d. As arm 302 moves back and forth (left and right in
FIG. 3) and side to side (into and out of the plane of FIG. 3), a
plurality of probes selected from 304a-d are lowered into
individual source wells 308 of well plate 306 to obtain a
sample.
[0062] Once a sample is picked up by one or more of the plurality
of probes 304a-d, the sample is moved through a conduit 310a-d in
communication with the corresponding probe selected from 304a-d
such that the conduit extends from autosampler XYZ arm 302 to the
particle analyzers in communication with the respective tube
310a-d. A particle analyzer 330, 332, 334, 336 comprises a flow
cell and a laser interrogation device. A laser interrogation device
examines individual samples flowing through a flow cell located
therein at a laser interrogation point. In between intaking sample
material from each of source wells 308, one or more of the
plurality of probes 304a-d is allowed to intake a fluid such as
air, thereby forming a gas separation between each adjacent sample.
The continuous fluid flow stream from each of the probes 304a-d and
through the respective tube 310a-d are communicated to a respective
individual particle analyzer 330, 332, 334, 336. The sample is
moved to a particle analyzer by a fluid movement device which may
be located before the probe, between the probe and the particle
analyzer or after the particle analyzer.
[0063] FIG. 4 illustrates an alternative embodiment of the
multi-sample particle analyzer apparatus 400 of the present
invention. Multi-sample particle analyzer apparatus 400 preferably
comprises conventional XYZ stage 402 on which is positioned a well
plate 406. The plurality of hollow probes 404a-d are stationary as
stage 402 moves back and forth (left and right in FIG. 4) and side
to side (into and out of the plane of FIG. 4), the stage 402 raises
and lower the well plate 406 to a plurality of probes 404a-d so
that individual samples from individual source wells 408 are
obtained of well plate 406. The probes may be positioned on one or
more arms (sometimes referred to herein as a bracket) with each arm
independently controlled to coordinate sampling of the sample
wells. The arms may be controlled by a computer with instructions
for moving the sample well plate and/or the probes.
[0064] Once a sample is picked up by one or more of the plurality
of probes 404a-d, the sample is moved through a tube 410a-d in
communication with the corresponding probe selected from 404a-d
such that the tube extends from the respective probe 404a-d into a
multi-channel fluid movement device 420 and then into the particle
analyzers in communication with the respective tube 210a-d.
However, the multi-channel fluid movement device 410a-d may be
located after the particle analyzer or before the sample probe. A
particle analyzer 430, 432, 434, 436 comprises a flow cell and a
laser interrogation device. A laser interrogation device examines
individual samples flowing through a flow cell located therein at a
laser interrogation point. In between intaking sample material from
each of source wells 408, one or more of the plurality of probes
404a-d is allowed to intake a fluid such as air, thereby forming a
gas separation between each adjacent sample. The continuous fluid
flow stream from each of the probes 404a-d and through the
respective tube 410a-d are communicated to a respective individual
particle analyzer 430, 432, 434, 436.
[0065] FIG. 5 illustrates an alternative embodiment of the
multi-sample particle analyzer apparatus 500 of the present
invention. Multi-sample particle analyzer apparatus 500 preferably
comprises conventional autosampler having adjustable XYZ
positioning arm 502 on which is positioned one or more hollow
probes 504a-d. As arm 502 moves back and forth (left and right in
FIG. 5) and side to side (into and out of the plane of FIG. 5), a
plurality of probes selected from 504a-d are lowered into
individual source wells 508a-g of well plate 506 to obtain a
separate sample for each probe.
[0066] Once a portion of a sample is withdrawn from a sample well
by one or more of the plurality of probes 504a-d, the sample is
moved through a conduit such as a tube 510a-d in communication with
the corresponding probe selected from 504a-d such that the tube
extends from autosampler XYZ arm 502 and into the particle
analyzers in communication with the respective tube 510a-d. The
particle analyzer 530, 532, 534, 536 may comprise a flow cell and a
laser interrogation device. A laser interrogation device examines
individual samples flowing past a laser interrogation point within
the particle analyzer. In between intaking sample material from
each of source wells 508, one or more of the plurality of probes
504a-d is allowed to intake a fluid such as air, thereby forming a
gas separation between each adjacent sample. The continuous fluid
flow stream from each of the probes 504a-d and through the
respective tube 510a-d are communicated to a respective individual
particle analyzer 530, 532, 534, 536.
[0067] FIG. 6 illustrates an alternative embodiment of the
multi-sample particle analyzer apparatus 600 of the present
invention. Multi-sample particle analyzer apparatus 600 preferably
comprises conventional XYZ stage 602 on which is positioned a well
plate 606. The plurality of hollow probes 604a-d are stationary as
stage 602 moves back and forth (left and right in FIG. 6) and side
to side (into and out of the plane of FIG. 6), the stage 602 raises
and lower the well plate 606 to a plurality of probes 604a-d so
that individual samples from individual source wells 608 are
obtained from well plate 606. Probes 604a-d may be fixed in space
relative to each other or may move relative to the stage 602 and
relative to the other probes 604a-d.
[0068] Once a sample is picked up by one or more of the plurality
of probes 604a-d, the sample is moved through a tube 610a-d in
communication with the corresponding probe selected from 604a-d
such that the tube extends from the respective probe 604a-d to the
particle analyzer to deliver the sample for analysis. A particle
analyzer 630, 632, 634, 636 may comprise a flow cell and a laser
interrogation device. A laser interrogation device examines
individual samples flowing within the particle analyzer by focusing
a beam of light at a laser interrogation point and detecting the
beam of laser light or alterations at an optical detector. In
between intaking sample material from each of source wells 608, one
or more of the plurality of probes 604a-d is allowed to intake a
fluid such as air, thereby forming a gas separation between each
adjacent sample. The continuous fluid flow stream from each of the
probes 604a-d and through the respective tube 610a-d are
communicated to a respective individual particle analyzer 630, 632,
634, 636.
[0069] FIG. 7 illustrates an alternative embodiment of the
multi-sample particle analyzer apparatus 700 of the present
invention. Multi-sample particle analyzer apparatus 700 preferably
comprises conventional autosampler having adjustable XYZ
positioning arm 702 on which is positioned a plurality of hollow
probes 704a-d. As arm 702 moves back and forth (left and right in
FIG. 7) and side to side (into and out of the plane of FIG. 7), a
plurality of probes selected from 704a-d are lowered into
individual source wells 708 of well plate 706 to obtain a
sample.
[0070] Once a sample is picked up by one or more of the plurality
of probes 704a-d, the sample is moved through a tube 710a-d in
communication with the corresponding probe selected from 704a-d
such that the tube extends from autosampler XYZ arm 702 and into a
multi-channel fluid movement device 720 and then into the particle
analyzer in communication with the respective tube 710a-d. However,
the fluid movement device may be located after the particle
analyzer 730 or before the probes. A plurality of samples are
communicated to a single multi-channel particle analyzer 730 which
will multiplex the samples to a single laser interrogation device
and analyze the samples simultaneously. A laser interrogation
device examines individual samples flowing through a flow cell
located within the particle analyzer at a laser interrogation
point. In between uptaking sample material from each of source
wells 708, one or more of the plurality of probes 704a-d is allowed
to uptake a fluid such as air, thereby forming a gas separation
between each adjacent sample. The continuous fluid flow stream from
each of the probes 704a-d and through the respective tube 710a-d
are communicated to a multi-channel particle analyzer 730.
[0071] FIG. 8 illustrates an alternative embodiment of the
multi-sample particle analyzer apparatus 800 of the present
invention. Multi-sample particle analyzer apparatus 800 preferably
comprises conventional XYZ stage 802 on which is positioned a well
plate 806. A plurality of probes 804a-d are stationary as stage 802
moves back and forth (left and right in FIG. 8) and side to side
(into and out of the plane of FIG. 8), the stage 802 raises and
lower the well plate 806 to the plurality of probes 804a-d so that
individual samples from individual source wells 808 of well plate
806 are sampled. The plurality of probes have an open first end and
an open second end apposite the first end. The open second end
connects to a conduit. The sample flows from the sample well to the
particle analyzer through the conduit. A conduit as used herein may
be a channel, a tube, a groove or other structure that guides the
fluid sample stream or fluid flow path to the particle
analyzer.
[0072] Once a sample is picked up by one or more of the plurality
of probes 804a-d, the sample is moved through a conduit 810a-d in
communication with the corresponding probe selected from 804a-d
such that the conduit extends from the respective probe 804a-d into
a multi-channel fluid movement device 820 and then into the
particle analyzers in communication with the respective conduit
810a-d. However, the multiple single-channel fluid movement devices
920a-d may be positioned after the particle analyzer or before the
probes. The plurality of samples are communicated to a single
multi-channel particle analyzer 830 which will multiplex the
samples to a single laser interrogation device and analyze the
samples simultaneously. In between uptaking sample material from
each of source wells 808, one or more of the plurality of probes
804a-d is allowed to intake a fluid such as air, thereby forming a
gas separation between each adjacent sample. The continuous fluid
flow stream from each of the probes 804a-d and through the
respective tube 810a-d are communicated to a multi-channel particle
analyzer 830.
[0073] FIG. 9 illustrates an alternative embodiment of the
multi-sample particle analyzer apparatus 900 of the present
invention. Multi-sample article analyzer apparatus 900 preferably
comprises conventional autosampler having adjustable XYZ
positioning arm 902 on which is positioned a plurality of hollow
probes 904a-d. As arm 902 moves back and forth (left and right in
FIG. 9) and side to side (into and out of the plane of FIG. 9), a
plurality of probes selected from 904a-d are lowered into
individual source wells 908 of well plate 906 to obtain a
sample.
[0074] Once a sample is picked up by one or more of the plurality
of probes 904a-d, the sample is moved through a tube 910a-d in
communication with the corresponding probe selected from 904a-d
such that the tube extends from autosampler XYZ arm 902 and into
multiple single-channel fluid movement devices 920a-d and then into
the particle analyzers in communication with the respective tube
910a-d. However, the multiple single-channel fluid movement devices
920a-d may be position after the particle analyzer or before the
probes. The fluid flow streams may be moved at different rates or
the same rates by the fluid movement device. A plurality of samples
are communicated to a single multi-channel particle analyzer 930
which will multiplex the samples to a single laser interrogation
device and analyze the samples simultaneously. A laser
interrogation device examines individual samples flowing through a
flow cell located therein at a laser interrogation point. In
between intaking sample material from each of source wells 908, one
or more of the plurality of probes 904a-d is allowed to intake a
fluid such as air, thereby forming a gas separation between each
adjacent sample. The continuous fluid flow stream from each of the
probes 904a-d and through the respective tube 910a-d are
communicated to a multi-channel particle analyzer 930.
[0075] FIG. 10 illustrates an alternative embodiment of the
multi-sample particle analyzer apparatus 1000 of the present
invention. Multi-sample particle analyzer apparatus 1000 preferably
comprises conventional XYZ stage 1002 on which is positioned a well
plate 1006. The plurality of hollow probes 1004a-d are stationary
as stage 1002 moves back and forth (left and right in FIG. 10) and
side to side (into and out of the plane of FIG. 10), the stage 1002
raises and lower the well plate 1006 to a plurality of probes
1004a-d so that individual samples from individual source wells
1008 are obtained of well plate 1006.
[0076] Once a sample is picked up by one or more of the plurality
of probes 1004a-d, the sample is moved through a tube 1010a-d in
communication with the corresponding probe selected from 1004a-d
such that the tube extends from the respective probe 1004a-d into a
multi-channel fluid movement device 1020 and then into the particle
analyzers in communication with the respective tube 210a-d.
However, the fluid movement device 1020a-d may be located after the
particle analyzer 1030. The plurality of samples are communicated
to a single multi-channel particle analyzer 1030 which will
multiplex the samples to a single laser interrogation device and
analyze the samples simultaneously. In between intaking sample
material from each of source wells 1008, one or more of the
plurality of probes 1004a-d is allowed to intake a fluid such as
air, thereby forming a gas separation between each adjacent sample.
The continuous fluid flow stream from each of the probes 1004a-d
and through the respective tube 1010a-d are communicated to a
multi-channel particle analyzer 1030.
[0077] FIG. 11 illustrates an alternative embodiment of the
multi-sample particle analyzer apparatus 1100 of the present
invention. Multi-sample particle analyzer apparatus 1100 preferably
comprises an autosampler having adjustable XYZ positioning arm 1102
on which is positioned a plurality of hollow probes 1104a-d. As arm
1102 moves back and forth (left and right in FIG. 11) and side to
side (into and out of the plane of FIG. 11), a plurality of probes
selected from 1104a-d are lowered into individual source wells 1108
of well plate 1106 to obtain a sample.
[0078] Once a sample is picked up by one or more of the plurality
of probes 1104a-d, the sample is moved through a tube 1110a-d in
communication with the corresponding probe selected from 1104a-d
such that the tube extends from autosampler XYZ arm 1102 and into
the particle analyzers in communication with the respective tube
1110a-d. The plurality of samples are communicated to a single
multi-channel particle analyzer 1130 which will multiplex the
samples to a single laser interrogation device and analyze the
samples simultaneously. A laser interrogation device interrogates a
sample flowing through a flow cell located therein at a laser
interrogation point. In between uptaking individual samples from a
second source well 1108a-g, a probe 1104a-d introduces a fluid such
as air between a first sample and a second sample, thereby forming
a gas separation between the first sample and the second sample to
form a continuous fluid flow stream to be analyzed. The continuous
fluid flow stream from each of the probes 1104a-d and through the
respective tube 1110a-d are communicated to a multi-channel
particle analyzer 1130.
[0079] FIG. 12 illustrates an alternative embodiment of the
multi-sample particle analyzer apparatus 1200 of the present
invention. Multi-sample particle analyzer apparatus 1200 preferably
comprises conventional XYZ stage 1202 on which is positioned a well
plate 1206. The plurality of hollow probes 1204a-d are stationary
as stage 1202 moves back and forth (left and right in FIG. 12) and
side to side (into and out of the plane of FIG. 12), the stage 1202
raises and lower the well plate 1206 to a plurality of probes
1204a-d so that individual samples from individual source wells
1208 are obtained of well plate 1206.
[0080] Once a sample is withdrawn by one or more of the plurality
of probes 1204a-d, the sample is moved through a tube 1210a-d in
communication with the corresponding probe selected from 1204a-d
such that the tube extends from the respective probe 1204a-d into
the particle analyzers in communication with the respective tube
1210a-d. The plurality of samples are communicated to a single
multi-channel particle analyzer 1230 which will multiplex the
samples to a single laser interrogation device and analyze the
samples simultaneously. A laser interrogation device examines
individual samples flowing through a flow cell located therein at a
laser interrogation point. In between intaking sample material from
each of source wells 1208, one or more of the plurality of probes
1204a-d is allowed to intake a fluid such as air, thereby forming a
gas separation between each adjacent sample. The continuous fluid
flow stream from each of the probes 1204a-d and through the
respective tube 1210a-d are communicated to a multi-channel
particle analyzer 1230.
[0081] FIG. 13 illustrates cytometry results of forward scatter vs.
side scatter with a gate around the particles aligned in the laser
beam according to one embodiment of the present invention.
[0082] FIG. 14 illustrates flow cytometry results using a fluid
means to move samples 1402, 1406, 1410, and 1414 naming particles
having a first fluorochrome as a fluorescence tag and four samples
1404, 1408, 1412, and 1416 having particles having a second
fluorochrome as a fluorescence tag. FIG. 14 is a graph of
Fluorescence vs. Time (1024 channels=60 seconds).
[0083] FIG. 15 illustrates an embodiment of the present invention
wherein multiple sample probes 1504a-d are positioned in a
plurality of sample source wells 1508a-d from a plurality of plates
1506a-d. The samples withdrawn from the plates by the probes are
moved to a particle analyzer (not shown) through conduits 1510a-d
connected to a respective probe 1508a-d. The plates may be
positioned on a platform that moves the plates in the XYZ
dimensions in space or the probes may move in the XYZ dimensions in
space relative to the plates, relative to each other or both.
[0084] The autosampler of the present invention may be any
conventional autosampler suitable for intaking samples from a well
plate or other sample source container. For example, an autosampler
as produced by TM the Gilson 223 liquid manager.
[0085] The XYZ stage of the present invention may be any XYZ stage
suitable for positioning a well plate.
[0086] The use of automation, for example a robotic plate loader,
in plate delivery and retrieval for the autosampler or XYZ stage
may allow automation of the overall screening process.
[0087] One preferred probe for the present invention is a 0.01 inch
ID, inch OD stainless steel needle compatible with HPLC ferrule
fittings. Similar probes with reinforced tubular sheaths are suited
for multiprobe sampling. The current embodiment of the invention
utilizes a Gilson interface module for bidirectional communication
between a computer and a probe manipulating arm or XYZ stage, and a
fluid movement device. Software designed using commercial
languages, such as C++, C#, Java, etc. may be used to control the
speed and distance of probe motions in all 3 dimensions, the
sensing of probe contact with liquid in a source well to assure
reproducible sample volumes, and the speed of the fluid movement
device. A computer or other known device may be used to control the
autosampler or XYZ stage to regulate sample size and bubble size by
varying the time that the probe is in a source well or above a
source well. Also, various sample handlers, XYZ stages, and sampler
handling systems that may be useful in the apparatus and method of
the present invention are well known in the art.
[0088] In order to reduce sample carryover a rinsing station or
device that may be attached to the autosampler to rinse the
autosampler probe between intakes of sample and/or buffer solution.
The rinsing fluid may be water, a mild detergent, or a solvent,
such as a solvent in which each of the particles in one or more of
the samples is dissolved. When the particles are merely suspended
in a suspension fluid, the rinsing fluid may be the same as the
suspension fluid.
[0089] Various conventional means may be employed to move the
sample through the system. For example, peristaltic pumps or
injection devices may be used with a particle analyzer such as a
flow cytometer or an acoustic cytometry apparatus for the present
invention. According to one embodiment, the peristaltic pump is a
multi channel or head pump that accommodates up to 4 separate
sample tubes leading to particle analyzers.
[0090] There are various types of tubing may be used for the fluid
flow path of the present invention, as long as the tubing may
function as high speed multi-sample tubing. When thin walled PVC
(polyvinyl chloride) tubing is used as the tubing for the present
invention, carryover between samples is substantially reduced
compared to conventional peristaltic tubing.
[0091] Various types of flow cytometers may be used with the
present invention. Commercially available flow cytometers from
Becton Dickinson (FACSCAIibur, FACSArray, FACSCantoll and LSRII),
Beckman Coulter (CyAn) and Accuri Cytometers (C6) have all been
proven compatible with embodiments of the current invention. The
use of the real-time software in conjunction with flow cytometer
controlling software may allow the samples from a given source well
to be rapidly re-checked during sampling and data analysis to prove
that "hits" from neighboring source wells do not arise from
cross-contamination, or to identify hits for additional treatment
or testing.
[0092] On-line data analysis may be used in the flow cytometer to
compare data between well plates and facilitate overall utility of
the data in conjunction with automation. Operation of the flow
cytometer at higher pressure generally increases the sample flow
rate and may, in some circumstances yield a higher throughput.
Also, operation of the flow cytometer with increased time
resolution in data software may allow resolution of samples at
higher throughput rates.
[0093] Both peristaltic pumps and air bubbles have been used in a
variety of detection devices with flowing samples. For example,
bubbles are commonly used in clinical instruments to separate
samples and the peristaltic pumps to move fluids. However, in flow
cytometry there is specific teaching against air bubbles with the
idea that, optimally, the bubbles should be removed from the sample
prior to injection into the flow cytometer. However, in the current
invention, carefully controlling the air bubble and using a
temporal separation of the specimens to eliminate the air bubble
from analysis, the introduction of air bubbles between samples has
proven to have little or no effect on the quality of results.
[0094] Using the flow cytometry apparatus of the present invention,
it has already been possible to move and analyze up to 40 samples
per minute in a single channel (consisting of a probe, tube,
peristaltic pump). In a preferred embodiment, utilizing multiple
probes, 80-400 samples can be moved and analyzed per minute.
[0095] Among the advantages of the flow cytometer apparatus of the
present invention is that it allows rapid sampling of small volumes
of sample. For example, a sample drawn into the fluid stream tubing
at a rate of about 0.3 ul/sec requires less than a 2 ul sample.
[0096] The throughput of the flow cytometry apparatus of the
present invention tends to be more affected by the behavior of the
autosampler rather than the characteristics of the fluid movement
device, the tubing or the flow cytometer. Thus, to the extent that
an autosampler can move more rapidly from source well to source
well, higher throughputs are achieved. Improved accuracy in volume
intake/delivery by the autosampler leads to smaller sample volumes
and improved throughputs.
[0097] In another embodiment of the present invention, a plurality
of samples are simultaneously picked up using a multichannel
autosampler comprising a plurality of sampling probes and a
plurality of sample delivery tubes. In this embodiment, a
peristaltic pump or other sample moving device moves the plurality
of samples in the delivery tubes through the pump simultaneously
using a plurality of pump heads or injection devices thus providing
a plurality of channels. By having a plurality of probes sampling a
plurality of wells of a microplate simultaneously, each channel is
delivered to a separate input port of a single particle analyzer or
separate particle analyzer to enable each channel to be analyzed in
parallel, thereby increasing sample throughput 100% for each
channel added.
[0098] Although the invention has been described in detail with
particular reference to these preferred embodiments, other
embodiments can achieve the same results. Variations and
modifications of the present invention will be obvious to those
skilled in the art and it is intended to cover in the appended
claims all such modifications and equivalents. For example, the arm
102 in FIG. 1 directs the movement of probe 104a independent of the
movement of probes 104b, 104c or 104d. For embodiments where there
are four probes illustrated, the invention is not limited thereto.
For example, the invention may include two or more probes each
attached to be a conduit leading to a separate particle analyzer or
a multi-channel particle analyzer. A means for moving the fluid can
be positioned before or after the particle analyzer or before the
probes. The entire disclosures of all references, applications,
patents, and publications cited above are hereby incorporated by
reference.
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