U.S. patent application number 14/667388 was filed with the patent office on 2015-10-29 for high throughput flow cytometry system and method.
The applicant listed for this patent is Nodality, Inc.. Invention is credited to James Cordiero, Todd M. Covey, Adam Palazzo, Norman Purvis, David Rosen.
Application Number | 20150309062 14/667388 |
Document ID | / |
Family ID | 42117880 |
Filed Date | 2015-10-29 |
United States Patent
Application |
20150309062 |
Kind Code |
A1 |
Covey; Todd M. ; et
al. |
October 29, 2015 |
HIGH THROUGHPUT FLOW CYTOMETRY SYSTEM AND METHOD
Abstract
The invention provides systems, compositions, kits and methods
for automated processing of biological samples and analysis using a
flow cytometer.
Inventors: |
Covey; Todd M.; (San Carlos,
CA) ; Palazzo; Adam; (San Francisco, CA) ;
Purvis; Norman; (Franklin, TN) ; Cordiero; James;
(Redwood City, CA) ; Rosen; David; (Mountain View,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nodality, Inc. |
So. San Francisco |
CA |
US |
|
|
Family ID: |
42117880 |
Appl. No.: |
14/667388 |
Filed: |
March 24, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12606869 |
Oct 27, 2009 |
9034257 |
|
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14667388 |
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61181211 |
May 26, 2009 |
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61162598 |
Mar 23, 2009 |
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61108803 |
Oct 27, 2008 |
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Current U.S.
Class: |
506/9 |
Current CPC
Class: |
G01N 2035/00752
20130101; G01N 35/0099 20130101; G01N 2035/00435 20130101; G01N
33/53 20130101; G01N 33/5008 20130101; G01N 35/00732 20130101; G01N
35/028 20130101; G01N 2035/0091 20130101; G01N 35/00722 20130101;
G01N 35/1095 20130101 |
International
Class: |
G01N 35/00 20060101
G01N035/00; G01N 35/02 20060101 G01N035/02; G01N 33/50 20060101
G01N033/50; G01N 35/10 20060101 G01N035/10 |
Claims
1-58. (canceled)
59. A method for automated or semi-automated processing of samples
and multiparametric analysis of the processed samples in a
cytometer, comprising: a. providing a system for processing samples
to produce processed samples prior to analyzing the processed
samples in the cytometer, comprising: 1. at least one microplate
and microplate holder; 2. a first reagent container containing a
first detectable binding element that is specific for an activation
state of a first activatable element, a second reagent container
containing a second detectable binding element that is specific for
a first cell surface marker, wherein the first and second
detectable binding elements are differentially detectable by the
cytometer, a third and fourth reagent containers containing a first
and second modulator, respectively, wherein the detectable binding
elements and the modulators are in liquid solution or suspension in
their respective reagent containers; 3. a dispensing head
configured to withdraw liquid from one of said reagent or sample
containers and dispense it to a well in said microplate, and/or to
withdraw liquid from a first well in the microplate and dispense it
to a second well in the same or different microplate; 4. a
microplate handling apparatus for moving said microplate, wherein:
i. said microplate handling apparatus is configured to position
said microplate near said dispensing head so that said dispensing
head can dispense liquid from a reagent container into a well in
said microplate and/or withdraw liquid from a well in said
microplate during the production of a processed sample or to
position said microplate so that it can be manually moved to be
near said dispensing head; and ii. said microplate handling
apparatus is configured to either relocate said microplate to said
cytometer for transfer of the processed sample to, and analysis of
the processed sample by, the cytometer, or to position said
microplate so that it may be relocated manually to said cytometer
for transfer of the processed sample to, and analysis of the
processed sample by the cytometer; b. providing a system for
analysis and management of the processed samples in step a,
comprising: 1. said cytometer, wherein the cytometer is configured
to differentially detect a plurality of differentially detectable
binding elements bound to activatable elements and cell surface
markers in single cells present in said samples; and 2. a computer
system including i. a processor, ii. a laboratory information
management system (LIMS) configured to track and record an
experiment, inventory management and experimental design and to
automate sample tracking from wherever the sample is initially
input into the automation system through to data analysis and
completion, and iii. a memory unit configured to control, and
operably connected to, said microplate holder, said tracking
mechanism, said microplate handling apparatus, said dispensing
head, and said cytometer, wherein the memory unit and the LIMS are
configured to communicate with the cytometer by instrument control
and image processing application software to inform the cytometer
as to plate layout, including the presence or absence of the first
or second differentially detectable binding elements, or
combination thereof, for each well in the plate, wherein there are
potentially at least 6 differentially detectable binding elements
and each well of the plate may contain one or more different
binding elements compared to other wells, as well as the presence
or absence of one or more modulators, with corresponding modulation
time, so as to direct proper analysis of each of the samples in
each well of the plate by the cytometer, in a manner compatible
with the cytometer software, wherein the computer system is
configured to consolidate elements of data or metadata to a single
file; c. withdrawing and dispensing liquids from said reagent
containers into one or more wells of the at least one microplate to
perform a protocol, wherein said protocol comprises: 1. preparing a
master modulator plate in an automated process wherein the first
modulator is removed from the third reagent container and dispensed
into a first well in the microplate and the second modulator is
removed from the fourth reagent container and dispensed into a
second well in the microplate; 2. providing a second microplate
comprising a first well and a second well, each of which contains a
biological sample comprising cells, and dispensing the first
modulator from the first well in the first microplate to the first
well in the second microplate and the second modulator from the
second well in the first microplate to the second well in the
second microplate, so that the modulators and the cells are in
contact, wherein said dispensing may be manual or automated or a
combination of manual and automated; and 3. determining an
activation level of at least said first activatable element in
single cells in said one or more samples, wherein said first
activatable element is an element in a pathway that is modulated by
the first modulator or the second modulator, by said cytometer,
wherein said activation level is determined by a process
comprising: contacting said at least one biological sample with the
first detectable binding element which is specific to an activation
state of said first activatable element, wherein said contacting
comprises: withdrawing said first detectable binding element from
said first reagent container and dispensing said first detectable
binding element to the well in the microplate containing said
sample such that said first detectable binding element contacts the
cells in said sample and binds to the first activatable element, if
present, and detecting said bound first detectable binding element
in said cells on a single cell basis in the cytometer; and 3.
determining a level of at least said first cell surface marker in
said one or more samples on a single cell basis by said cytometer,
wherein said first cell surface marker level is determined by a
process comprising: contacting said at least one biological sample
with the second detectable binding element which is specific to the
first cell surface marker, wherein said contacting comprises:
withdrawing said second detectable binding element from said second
reagent container and dispensing said second detectable binding
element to the well in the microplate containing said sample such
that said second detectable binding element contacts the cells in
said sample and binds to said first cell surface marker, if
present, and detecting said bound second detectable binding element
in said cells on a single cell basis in the cytometer.
60. The method of claim 59 wherein the automated process of c.1
comprises (a) initiating a liquid handler program using automated
software; (b) system requesting an appropriate stock modulator to
be loaded into automated system; and (c) automated system
automatically performs modulator dilutions to create working
solutions for the first modulator in the third reagent container
and the second modulator in the fourth reagent container, according
to liquid handler program.
61. The method of claim 59 wherein said samples comprise intact
viable cells and wherein said pre-processing comprises contacting
said intact viable cells with the modulator for a predetermined
time, and wherein the contacting of step c.2 further comprises
contacting the cells and the modulator in such a way that the
sample remains viable during the contacting for the predetermined
time.
62. The method of claim 61 wherein the computer system is
configured to control the contacting of the cells with the
modulator for the predetermined time.
63. The method of claim 62 wherein the computer system controls the
contacting of the cells with modulator for the predetermined time
by controlling a process comprising fixing of the sample after
treatment with the modulator.
64. The method of claim 63 wherein said process comprising fixing
of the sample after treatment with the modulator comprises
withdrawing the fixative from a reagent container or containers and
dispensing the fixative to the wells of the at least one microplate
containing the sample, or withdrawing said sample from a sample
container and dispensing the sample to wells of the at least one
microplate containing the fixative, such that the fixative contacts
the cells of the sample.
65. The method of claim 64 wherein the computer system controls the
contacting of the cells with fixative for a predetermined time.
66. The method of claim 59 wherein the system further comprises at
least one of a fifth reagent container containing a third
detectable binding element specific to an activation state of a
second activatable element, a sixth reagent container containing a
fourth detectable binding element specific to an activation state
of a third activatable element, or a seventh reagent container
containing a fifth detectable binding element specific to an
activation state of a fourth activatable element, wherein the
third, fourth, and fifth detectable binding elements are
differentially detectable from each other and from the first and
second binding elements, and wherein each detectable binding
element is contacted with cells in sample in at least one well of
the at least one microplate and detected by the cytometer.
67. The method of claim 66 wherein the system comprises at least
two of the fifth, sixth, and seventh reagent containers and their
respective detectable binding elements.
68. The method of claim 67 wherein the system further comprises a
reagent container containing a third modulator and wherein, in step
c.1, the third modulator is removed from its reagent container and
dispensed into a third well of the master modulator plate.
69. The method of claim 68 wherein the system further comprises a
reagent container containing a fourth modulator and wherein, in
step c.1, the fourth modulator is removed from its reagent
container and dispensed into a fourth well of the master modulator
plate.
70. The method of claim 69 wherein the system further comprises a
reagent container containing a fifth modulator and wherein, in step
c.1, the fifth modulator is removed from its reagent container and
dispensed into a fifth well of the master modulator plate.
71. The method of claim 70 wherein the first, second, third,
fourth, and fifth modulators are different from each other and the
first, second, third, fourth, and fifth wells are different from
each other.
72. The method of claim 59 wherein the cytometer is a flow
cytometer.
73. The method of claim 59 wherein the cytometer is a mass
cytometer.
74. The method of claim 59 wherein the single file is not
duplicated.
75. A method for automated or semi-automated processing of samples
and analysis of the processed samples by a cytometer, comprising:
a. providing a system for processing said samples prior to their
analysis in a cytometer, comprising: 1. at least one or more
microplates and microplate holder; 2. a first reagent container
containing a first detectable binding element that is specific for
an activation state of a first activatable element, a second
reagent container containing a second detectable binding element
that is specific for a first cell surface marker, wherein the first
and second detectable binding elements are differentially
detectable, and either a third reagent container containing a first
modulator or a sample container containing a biological sample
comprising cells, or both, wherein the modulator, the detectable
binding elements, and the biological sample comprising cells are in
liquid solution or suspension in their respective reagent
containers; 3. a dispensing head configured to withdraw liquid from
one of said reagent or sample containers and dispense it to a well
in said microplate and/or to withdraw liquid from a first well in
the microplate and dispense it to a second well in the same or
different microplate; 4. a microplate handling apparatus for moving
said microplate, wherein; i. said microplate handling apparatus is
configured to position said microplate near said dispensing head so
that said dispensing head can dispense liquid from a reagent
container into a well in said microplate and/or withdraw liquid
from a well in said microplate during the production of a processed
sample or to position said microplate so that it can be manually
moved to be near said dispensing head; and ii. said microplate
handling apparatus is configured to either relocate said microplate
to said cytometer for transfer of the processed sample to, and
analysis of the processed sample by, the cytometer, or to position
said microplate so that it may be relocated manually to said
cytometer for transfer of the processed sample to, and analysis of
the processed sample by the cytometer; b. providing a system for,
analysis and management of the processed samples in step a,
comprising: 1. said cytometer, wherein the cytometer is configured
to differentially detect a plurality of differentially detectable
binding elements bound to activatable elements and cell surface
markers in single cells present in said samples; 2. a computer
system, wherein said computer system comprises i. a processor, ii a
LIMS configured to track and record an experiment design and to
automate sample tracking from wherever the sample is initially
input into said automation process and analysis through to data
analysis and completion, and iii. a memory unit configured to
control, and operably connected to, said microplate holder, said
barcode reader, said microplate handling apparatus, the timing of
said microplate handling steps, said dispensing head, and said
cytometer, wherein the memory unit and the LIMS are configured to
communicate with the cytometer by instrument control and image
processing application software to inform the cytometer as to plate
layout, including the presence or absence of the first or second
differentially detectable binding elements, or combination thereof,
for each well in the plate, wherein there are potentially at least
6 differentially detectable binding elements and each well of the
plate may contain one or more different binding elements compared
to other wells, as well as the presence or absence of one or more
modulators, with corresponding modulation time, so as to direct
proper analysis of each of the samples in each well of the plate by
the cytometer, in a manner compatible with the cytometer software,
wherein the computer system is configured to consolidate elements
of data or metadata to a single file; c. withdrawing and dispensing
liquids from said reagent containers into one or more wells of the
at least one microplate to perform a protocol, wherein said
protocol comprises: 1. either: i. providing the at least one
microplate, one or more of whose wells contains biological sample
comprising cells, and dispensing modulator from said third reagent
container into the wells so that the modulator and the cells are in
contact; or ii. providing the at least one microplate, one or more
of whose wells contains a modulator and dispensing at least one
biological sample comprising cells from said sample container into
the wells so that the modulator and the cells are in contact; or
iii. providing the at least one microplate and dispensing at least
one biological sample from the sample container into one or more
wells of the microplate and dispensing modulator from the third
reagent container into one or more of the wells of the microplate,
so that the modulator and the cells are in contact; and 2.
contacting said samples with a fixative, wherein said contacting is
performed by automation at a time determined by the LIMs, and a
permeablization reagent; 3. determining an activation level of at
least said first activatable element in single cells in said one or
more samples wherein said first activatable element is an element
in a pathway that is modulated by the modulator, by said cytometer,
wherein said activation level is determined by a process
comprising: contacting said at least one biological sample with the
first detectable binding element which is specific to an activation
state of said first activatable element, wherein said contacting
comprises: withdrawing said first detectable binding element from
said first reagent container and dispensing said first detectable
binding element to the well in the microplate containing said
sample such that said first detectable binding element contacts the
cells in said sample and binds to the first activatable element, if
present, and detecting said bound first detectable binding element
in said cells on a single cell basis in the cytometer; 4.
determining a level of at least said first cell surface marker in
said one or more samples on a single cell basis by said cytometer,
wherein said first cell surface marker level is determined by a
process comprising: contacting said at least one biological sample
with the second detectable binding element which is specific to the
first cell surface marker, wherein said contacting comprises:
withdrawing said second detectable binding element from said second
reagent container and dispensing said second detectable binding
element to the well in the microplate containing said sample such
that said second detectable binding element contacts the cells in
said sample and binds to said first cell surface marker, if
present, and detecting said bound second detectable binding element
in said cells on a single cell basis in the cytometer; d. wherein
the LIMS software is configured to direct experimental design,
plate layout, sample tracking, and inventory management and one or
more of the following purposes from the group consisting of
instrument alignment; correct connections; motor operations; timing
for sample handling; and data analysis.
76. The method of claim 75, wherein the steps in the protocol are
conducted in islands of automation.
77. The method of claim 75, wherein one or more components of the
system are in a thermo regulating component.
78. The method of claim 75, wherein the microplate handling
apparatus comprises a robotic arm, and further comprising moving
the at least one microplate using the robotic arm.
79. A method for processing of samples and analysis of the
processed samples in a cytometer, comprising: (i) preparing a
master modulator microplate, wherein the master modulator plate
comprises at least a first well or tube containing a first
modulator at a first concentration and a second well or tube
containing a second modulator at a second concentration, each of
which contains one of a plurality of modulators, each modulator
being present at a predetermined concentration for that modulator,
comprising (a) initiate a liquid handler program using automated
software; (b) loading stock modulator for each well into automated
system according to a system request, wherein the loading may be
automated or manual; (c) automatically performing modulator
dilutions by the automated system to create working solutions in
working solution containers according to liquid handler program;
(d) automatically aliquoting working solutions into master
modulator plate by automated system, thus preparing a master
modulator plate according to liquid handler program; (ii) providing
a sample microplate, comprising a first well and a second well,
each of which contains a biological sample comprising cells, and
dispensing the first modulator from the first well in the master
modulator microplate to the first well in the sample microplate and
the second modulator from the second well in the master modulator
microplate to the second well in the sample microplate, so that the
modulators and the cells are in contact, wherein said dispensing
may be manual or automated or a combination of manual and
automated; (iii) adding fixative to the first well of the sample
microplate at a first time after modulator and cells come in
contact and to the second well of the sample microplate at a second
time after modulator and cells come in contact; and (iv) analyzing
single cells from the first well in a cytometer for the presence of
a first activatable element and analyzing cells from the second
well in the cytometer for the presence of a second activatable
element.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 12/606,869, filed Oct. 27, 2009, which claims priority to U.S.
Provisional Applications Nos. 61/108,803 filed Oct. 27, 2008,
61/162,598 filed Mar. 23, 2009, and 61/181,211 filed May 26, 2009.
Each of these applications is hereby expressly incorporated by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Cell preparation for analysis using a flow cytometer is
commonly used for monitoring progression of disease, monitoring
therapy, and for screening compounds that may be used to treat a
disease.
[0003] Current techniques offer little in the way of automation for
preparing samples and analyzing them using flow cytometry. Current
protocols for cell preparation are mostly performed by human
operators. This means that sample throughput is limited to what a
single person or team of people can accomplish in a given amount of
time.
[0004] Accordingly, there is a need for a process that has the
advantage of generating less labor intensive and more reproducible
results. It is advantageous to generate reliable data from highly
controlled complex laboratory automation for the study of cell
signaling and for drug screening either in the form of complete
walk-away automation or with user intervention at defined points. A
highly controlled automated process can increase throughput, reduce
the potential for introducing variability into the results, and
ensure that meaningful results are generated.
SUMMARY OF THE INVENTION
[0005] The present invention relates to the processing of cells for
analysis. More specifically, the present invention relates to a
high throughput process and system for analyzing cells using a flow
cytometer.
[0006] In accordance with one embodiment described herein, a system
is described that facilitates automated and high-throughput
processing of cell samples for flow cytometry. In some embodiments,
the invention describes a system that facilitates automated and
high-throughput processing of cell samples for phosphoflow
cytometry. In some embodiments, the invention describes a system
that facilitates automated and high-throughput processing of cell
samples for analyzing receptor function (e.g. drug transporter)
using flow cytometry. This system includes one or more robotic
liquid handling instruments capable of performing sample transfer,
reagent addition, mixing, aspiration, incubation, and
centrifugation on a plurality of microplates or cartridges
containing biological materials. One embodiment is comprised of
multiple automation systems that perform fewer functions and may
require some human intervention. One embodiment is a fully
automated system.
[0007] Particular embodiments are integrated systems for performing
flow cytometry assays, acquisition, compilation, storage, and
analysis of data. These systems may include one or more robotic
liquid handling instruments with or without barcode scanners,
operably linked to one or more digital computers with software
including an instruction set, an input device, and a user interface
to input plate data.
[0008] In a particular embodiment, automated and semi-automated
instruments, as well as peripheral equipment, exchange sample
information and other relevant processing data with a laboratory
integrated management system (LIMS). In a particular embodiment,
applications and instruments may communicate with, or receive
information from LIMS.
[0009] In a particular embodiment, the cytometer is part of the
automated platform which may contain a liquid handler, automated
centrifuge (V-spin), incubator, Hypercyte, cytometer controlled by
a scheduling software, and a pathway analysis tool at the back end.
In a particular embodiment, the system be an automated approach to
pathway profiling starting from sample (ie, blood, marrow, purified
cells, cell lines) and ending with a full signaling pathway
work-up. In a particular embodiment, the system may be an automated
or semi-automated instrument that may be operably linked to a
computational device equipped with user input and output
features.
[0010] In accordance with a particular embodiment, a method is
described for automatic retrieval of a plurality of microplates, as
well as automatic sample transfer, reagent addition, mixing,
aspiration, incubation, and centrifugation of those microplates.
The method may include the steps of retrieving a selected
microplate from the plate holder, performing specified steps of the
assay on that plate in a controlled and timed fashion, and then the
final step of returning the completed microplate to the plate
holder.
[0011] Applications of the present invention may include probing
the various pathways by analyzing responses to a host of stimuli.
Applications may also include using the system in combination with
a compound library to assess the inhibition or enhancement of
various signaling nodes, assessing expression level and
functionality of various drug transporters, or probing the
signaling differences among various cancer cell lines (ie, NCI 60
cell lines). Examples of drug transporters include, but are not
limited to, glycoprotein (MDR1), MDR-associated protein and breast
cancer resistance protein. Particular embodiments may be used to
screen the supernatants from hybridomas in order to pick the best
antibody secreting cell clone. For instance, one could screen for a
neutralizing or agonistic antibody to a growth factor receptor by
looking for the supernatant that best augments the signaling
downstream of the growth factor receptor. In another embodiment one
could use the platform to select the best cell clone secreting any
protein that has a biological effect that could be measured by its
downstream effect on signaling.
INCORPORATION BY REFERENCE
[0012] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0014] FIG. 1 illustrates a partially automated system that
facilitates high-throughput processing of cell samples for
phosphoflow cytometry.
[0015] FIG. 2 illustrates a partially automated system that
facilitates high-throughput processing of cell samples for
phosphoflow cytometry.
[0016] FIG. 3A illustrates a fully automated system that
facilitates high-throughput processing of cell samples for
phosphoflow cytometry.
[0017] FIG. 3B illustrates a fully automated system that
facilitates high-throughput processing of cell samples for
phosphoflow cytometry.
[0018] FIG. 4 illustrates an instrument for processing samples.
[0019] FIG. 5 illustrates an example method for sample
preparation.
[0020] FIG. 6 illustrates an example apoptosis assays method.
[0021] FIG. 7 illustrates an example cell stimulation assays
method.
[0022] FIG. 8 illustrates an example kinetic timepoint assay
method.
[0023] FIG. 9 illustrates an example efflux assay method.
DETAILED DESCRIPTION OF THE INVENTION
[0024] High throughput flow cytometry system and method has many
preferred embodiments and relies on many patents, applications and
other references for details known to those of the art. Therefore,
when a patent, application, or other reference is cited or repeated
below, it should be understood that it is incorporated by reference
in its entirety for all purposes as well as for the proposition
that is recited.
[0025] As used in this application, the singular form "a," "an,"
and "the" include plural references unless the context clearly
dictates otherwise. For example, the term "an agent" includes a
plurality of agents, including mixtures thereof.
[0026] Throughout this disclosure, various aspects of this
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible sub-ranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed sub-ranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0027] Particular embodiments of the high throughput flow cytometry
system are designed to automate and improve the throughput of flow
cytometry systems, especially those that analyze cell signaling
networks and cell receptor function. Particular embodiments of the
automated system provide a highly controlled platform to screen
large numbers of samples that may then be analyzed using
multiparametric flow cytometry. Particular embodiments of the
automated system may be used in research and development,
diagnostic testing, clinical trials, testing cell lines, drug
screening and drug discovery applications. Particular embodiments
of the automated system may be designed to reduce processing cost,
reduce processing variability, or improve data integrity.
[0028] Particular embodiments comprise fully integrated, highly
controlled automated sample processing. In a particular embodiment,
a robotic arm may operate around a variety of instruments that are
involved in sample preparation for the analysis. Those instruments
may include a flow cytometer, a sample acquisition device (Hypercyt
for example), microcentrifuge tubes holding cell samples,
microplates holding cell samples, centrifuges, reagent containers,
temperature controlled incubators, thermal cyclers, temperature
controlled refrigeration units, liquid handling devices, automated
pipetting devices, automated dispensers, plate sealers, plate
piercers, barcode scanners, plate holders and may be operated by a
computer running the appropriate software using the appropriate
graphical user interface (GUI), which may be integrated into a
laboratory information management system (LIMS). In a particular
embodiment, the robotic arm may remove microplates containing cell
samples from an incubator or plate holder, the plates would be
placed on a deck as a working surface to interact with other
instruments and to accept the appropriate reagents. A liquid
handling or micropetting assembly may pull reagents from a plate
and add them to the cells (or vice versa). Scheduling software may
measure the appropriate incubation times and stop the reaction when
necessary. The cycles may be repeated as required for the
stimulation and preparation of the cells for flow cytometer
analysis and the cells may be centrifuged and washed to prepare a
pellet. The robotic arm may take the cell sample and pass to a
refrigeration unit for preservation or may subject it to immediate
analysis.
[0029] Particular embodiments comprise islands of automation
requiring human intervention to move samples from one process to
the next. In a particular embodiment a plurality of components are
involved with sample preparation for sample analysis. Those
components may include a flow cytometer, a sample acquisition
device (Hypercyt for example), microcentrifuge tubes holding cell
samples, microplates holding cell samples, centrifuges, reagent
containers, temperature controlled incubators, thermal cyclers,
temperature controlled refrigeration units, liquid handling
devices, automated pipetting devices, automated dispensers, plate
sealers, plate piercers, barcode scanners, plate holders and each
instrument may be operated by computers running the appropriate
software using the appropriate graphical user interface (GUI),
which may be integrated into a laboratory information management
system (LIMS).
BACKGROUND INFORMATION
[0030] Particular embodiments of the high throughput flow cytometry
system and method incorporate information disclosed in other
applications and texts. The following patents and other
publications are hereby incorporated by reference in their
entireties: Haskell et al, Cancer Treatment, 5.sup.th Ed., W.B.
Saunders and Co., 2001; Alberts et al., The Cell, 4.sup.th Ed.,
Garland Science, 2002; Vogelstein and Kinzler, The Genetic Basis of
Human Cancer, 2d Ed., McGraw Hill, 2002; Michael, Biochemical
Pathways, John Wiley and Sons, 1999; Weinberg, The Biology of
Cancer, 2007; Immunobiology, Janeway et al. 7.sup.th Ed., Garland,
and Leroith and Bondy, Growth Factors and Cytokines in Health and
Disease, A Multi Volume Treatise, Volumes 1A and 1B, Growth
Factors, 1996. Patents and applications that are also incorporated
by reference include U.S. Pat. Nos. 7,314,595, 7,368,084, 7,381535
and 7,393,656 and U.S. patent application Ser. Nos. 10/193,462;
11/655,785; 11/655,789; 11/655,821; 11/338,957, 61/048,886;
61/048,920; 61/048,657; 61/055,362; 61/079,766; 61/085,789;
61/079,551; 61/079,537; 61/079,579; 61/087,555; 61/120,320; and
61/153,627. Relevant articles include High-content single-cell drug
screening with phosphospecific flow cytometry, Krutzik et al.,
Nature Chemical Biology, 23 Dec. 2007; Irish et al., FLt3 ligand
Y591 duplication and Bcl-2 over expression are detected in acute
myeloid leukemia cells with high levels of phosphorylated wild-type
p53, Neoplasia, 2007, Irish et al. Mapping normal and cancer cell
signaling networks: towards single-cell proteomics, Nature, Vol. 6
146-155, 2006; and Irish et al., Single cell profiling of
potentiated phospho-protein networks in cancer cells, Cell, Vol.
118, 1-20 Jul. 23, 2004; Schulz, K. R., et al., Single-cell
phospho-protein analysis by flow cytometry, Curr Protoc Immunol,
2007, 78:8 8.17.1-20; Krutzik, P. O., et al., Coordinate analysis
of murine immune cell surface markers and intracellular
phosphoproteins by flow cytometry, J Immunol 2005 Aug. 15;
175(4):2357-65; Krutzik, P. O., et al., Characterization of the
murine immunological signaling network with phosphospecific flow
cytometry, J Immunol 2005 Aug. 15; 175(4):2366-73; Shulz et al.,
Current Protocols in Immunology 2007, 78:8.17.1-20; Stelzer et al.
Use of Multiparameter Flow Cytometry and Immunophenotyping for the
Diagnosis and Classification of Acute Myeloid Leukemia,
Immunophenotyping, Wiley, 2000; and Krutzik, P. O. and Nolan, G.
P., Intracellular phospho-protein staining techniques for flow
cytometry: monitoring single cell signaling events, Cytometry A.
2003 October; 55(2):61-70; Hanahan D., Weinberg, The Hallmarks of
Cancer, CELL, 2000 Jan. 7; 100(1) 57-70; Krutzik et al, High
content single cell drug screening with phosphospecific flow
cytometry, Nat Chem Biol. 2008 February; 4(2):132-42. Experimental
and process protocols and other helpful information can be found at
http:/proteomices.stanford.edu. Articles and other references that
may be cited below are also incorporated by reference in their
entireties for all purposes. Some commercial reagents, protocols,
software and instruments that are useful in particular embodiments
of the present invention are available at the Becton Dickinson
Website http://www.bdbiosciences.com/features/products/, and the
Beckman Coulter website,
http://www.beckmancoulter.com/Default.asp?bhfv=7.
[0031] In addition to the references cited above, the practice of
the present invention may employ, unless otherwise indicated,
conventional techniques and descriptions of flow cytometry, organic
chemistry, polymer technology, molecular biology (including
recombinant techniques), cell biology, cancer biology,
biochemistry, and immunology, which are within the skill of the
art. Such conventional techniques can be found in articles,
patents, commercial websites, as well as other sources as
referenced above. Other conventional techniques can be shown in
standard laboratory manuals such as those recited above and also
including Genome Analysis: A Laboratory Manual Series (Vols. I-IV),
Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual,
PCR Primer: A Laboratory Manual, and Molecular Cloning: A
Laboratory Manual (all from Cold Spring Harbor Laboratory Press),
Stryer, L. (1995) Biochemistry (4.sup.th Ed.) Freeman, N.Y., Gait,
"Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press,
London, Nelson and Cox (2000), Lehninger, Principles of
Biochemistry 3.sup.rd Ed., W.H. Freeman Pub., New York, N.Y. and
Berg et al. (2002) Biochemistry, 5.sup.th Ed., W.H. Freeman Pub.,
New York, N.Y., all of which are herein incorporated in their
entirety by reference for all purposes.
[0032] Use of Computers
[0033] Particular embodiments of high throughput flow cytometry
system may be implemented in a localized or distributed computing
environment. For example, in a particular embodiment featuring a
localized computing environment, a flow cytometry system may be
operably linked to a computational device equipped with user input
and output features. In a distributed environment, the methods may
be implemented on a single computer, a computer with multiple
processes or, alternatively, on multiple computers. The computers
may be linked, e.g., through a shared bus, but more commonly, the
computer(s) are nodes on a network. The network may be generalized
or dedicated, at a local level or distributed over a wide
geographic area. In particular embodiments, the computers may be
components of an intranet or an internet. See U.S. Patent
Application No. 61/048,657 and U.S. Pat. No. 7,314,595 which are
incorporated by reference in its entirety for details of some
computer hardware that may be useful in the present invention.
[0034] Particular embodiments also contemplate the use of a
computer which may operate various instrumentation components,
liquid handling or micropipetting equipment, analysis instruments
or analysis software. The computer may be any type of computer
platform such as a workstation, a personal computer, a server, or
any other present or future computer. The computer typically
includes known components such as a processor, an operating system,
system memory, memory storage devices, and input-output
controllers, input-output devices, and display devices. Display
devices may include display devices that provides visual
information, this information typically may be logically and/or
physically organized as an array of pixels. A Graphical the user
interface (GUI) controller may also be included that may comprise
any of a variety of known or future software programs for providing
graphical input and output interfaces such as for instance GUI's.
For example, GUI's may provide one or more graphical
representations to a the user, and also be enabled to process the
user inputs via GUI's using means of selection or input known to
those of ordinary skill in the related art.
[0035] It will be understood by those of ordinary skill in the
relevant art that there are many possible configurations of the
components of a computer and that some components that may
typically be included in a computer are not shown, such as cache
memory, a data backup unit, and many other devices. The processor
may be a commercially available processor such as an Itanium.RTM.
or Pentium.RTM. processor made by Intel Corporation, a SPARC.RTM.
processor made by Sun Microsystems, an Athalon.TM. or Opteron.TM.
processor made by AMD corporation, or it may be one of other
processors that are or will become available. Some embodiments of
the processor may also include what are referred to as Multi-core
processors and/or be enabled to employ parallel processing
technology in a single or multi-core configuration. For example, a
multi-core architecture typically comprises two or more processor
"execution cores". In the present example each execution core may
perform as an independent processor that enables parallel execution
of multiple threads. In addition, those of ordinary skill in the
related will appreciate that the processor may be configured in
what is generally referred to as 32 or 64 bit architectures, or
other architectural configurations now known or that may be
developed in the future.
[0036] The processor executes an operating system, which may be,
for example, a Windows.RTM.-type operating system (such as
Windows.RTM. XP or Windows.RTM. Vista) from the Microsoft
Corporation; the Mac OS X operating system from Apple Computer
Corp. (such as 7.5 Mac OS X v10.4 "Tiger" or 7.6 Mac OS X v10.5
"Leopard" operating systems); a Unix.RTM. or Linux-type operating
system available from many vendors or what is referred to as an
open source; another or a future operating system; or some
combination thereof. The operating system interfaces with firmware
and hardware in a well-known manner, and facilitates processor in
coordinating and executing the functions of various computer
programs that may be written in a variety of programming languages.
The operating system, typically in cooperation with the processor,
coordinates and executes functions of the other components of
computer. The operating system also provides scheduling,
input-output control, file and data management, memory management,
and communication control and related services, all in accordance
with known techniques.
[0037] The system memory may be any of a variety of known or future
memory storage devices. Examples include any commonly available
random access memory (RAM), magnetic medium such as a resident hard
disk or tape, an optical medium such as a read and write compact
disc, or other memory storage device. Memory storage devices may be
any of a variety of known or future devices, including a compact
disk drive, a tape drive, a removable hard disk drive, USB or flash
drive, or a diskette drive. Such types of memory storage devices
typically read from, and/or write to, a program storage medium (not
shown) such as, respectively, a compact disk, magnetic tape,
removable hard disk, USB or flash drive, or floppy diskette. Any of
these program storage media, or others now in use or that may later
be developed, may be considered a computer program product. As will
be appreciated, these program storage media typically store a
computer software program and/or data. Computer software
applications, also called computer control logic, typically are
stored in system memory and/or the program storage device used in
conjunction with memory storage device.
[0038] Input-output controllers could include any of a variety of
known devices for accepting and processing information from a user,
whether a human or a machine, whether local or remote. Such devices
include, for example, modern cards, wireless cards, network
interface cards, sound cards, or other types of controllers for any
of a variety of known input devices. Output controllers of
input-output controllers could include controllers for any of a
variety of known display devices for presenting information to a
user, whether a human or a machine, whether local or remote. In a
particular embodiment, the functional elements of computer may
communicate with each other via system bus. Some of these
communications may be accomplished in alternative embodiments using
network or other types of remote communications.
[0039] In a particular embodiment, the computer implemented methods
and/or systems may be operated locally on a computer, on a network
server accessed through one or more terminals, or through one or
more machines accessed through an intranet or internet. The method
may also be implemented through a computer readable medium storing
a computer program implementing the method's functionality.
[0040] In particular embodiments, a computer application product
may comprise a computer-readable medium having control logic
(computer software program, including program code) stored therein.
The control logic, when executed by a processor, causes the
processor to perform functions described herein. In particular
embodiments, some functions may be primarily implemented in
hardware using a hardware state machine. Implementation of the
hardware state machine so as to perform the functions described
herein will be apparent to those skilled in the relevant arts.
[0041] As will be evident to those skilled in the relevant art, an
instrument control and image processing application, such as for
instance an implementation of instrument control and image
processing applications, if implemented in software, may be loaded
into and executed from system memory and/or memory storage device.
All or portions of the instrument control and image processing
applications may also reside in a read-only memory or similar
device of memory storage device, such devices not requiring that
the instrument control and image processing applications first be
loaded through input-output controllers. It will be understood by
those skilled in the relevant art that the instrument control and
image processing applications, or portions of it, may be loaded by
processor in a known manner into system memory, or cache memory
(not shown), or both, as advantageous for execution. Library files,
calibration data, experiment data, and internet client data may be
stored in system memory. For example, experiment data could include
data related to one or more experiments or assays such as
excitation wavelength ranges, emission wavelength ranges,
extinction coefficients and/or associated excitation power level
values, or other values associated with one or more fluorescent
labels. Additionally, internet client may include an application
enabled to accesses a remote service on another computer using a
network that may for instance comprise what are generally referred
to as "Web Browsers". In the present example some commonly employed
web browsers include Microsoft.RTM. Internet Explorer 7 with SPI
available from Microsoft Corporation, Mozilla Firefox.RTM. 1.5 from
the Mozilla Corporation, Safari 2.0 from Apple Computer Corp., or
other type of web browser currently known in the art or to be
developed in the future. In particular embodiments, the internet
client may include, or could be an element of, specialized software
applications enabled to access remote information via a network
such as network.
[0042] The network may include one or more of the many various
types of networks well known to those of ordinary skill in the art.
For example, the network may include a local or wide area network
that employs what is commonly referred to as a TCP/IP protocol
suite to communicate, that may include a network comprising a
worldwide system of interconnected computer networks that is
commonly referred to as the internet, or could also include various
intranet architectures. Those of ordinary skill in the related arts
will also appreciate that some the users in networked environments
may prefer to employ what are generally referred to as "firewalls"
(also sometimes referred to as Packet Filters, or Border Protection
Devices) to control information traffic to and from hardware and/or
software systems. For example, firewalls may comprise hardware or
software elements or some combination thereof and are typically
designed to enforce security policies put in place by the users,
such as for instance network administrators, etc.
[0043] Instrument control and image processing applications may
comprise any of a variety of known or future image processing
applications. Typically, particular embodiments of applications may
be loaded into system memory and/or memory storage device.
[0044] In particular embodiments, applications may be stored on a
server or computer that may be accessed located locally or remotely
and communicate with one or more additional servers and/or one or
more other computers/workstations or instruments. In a particular
embodiment, applications may communicate with, and receive
instruction or information from, or control one or more elements or
processes of one or more servers, one or more workstations, and one
or more instruments or system components.
[0045] In particular embodiments, automated and semi-automated
instruments as well as peripheral equipment exchange sample
information with a laboratory integrated management system (LIMS)
or laboratory information system (LIS). In a particular embodiment,
applications and components may communicate with, or receive
information from LIMS or LIS. LIMS or LIS is typically associated
with a computer workstation containing software applications, GUI,
or instruments, and automates sample tracking from initial input
into the system to data analysis. For example, see U.S. Patent
Application No. 61/079,537.
[0046] Those skilled in the art will appreciate that applications
may be stored for execution on any compatible computer system, such
as computer, or any compatible computer readable tangible medium.
Embodiments of applications may advantageously provide what is
referred to as a modular interface for one or more computers or
workstations and one or more servers, as well as one or more
instruments. The term "modular" as used herein generally refers to
elements that may be integrated to and interact with a core element
in order to provide a flexible, updateable, and customizable
platform. For example, as will be described in greater detail below
applications may comprise a "core" software element enabled to
communicate and perform primary functions necessary for any
instrument control and image processing application. Such primary
functionality may include communication over various network
architectures, or data processing functions such as processing raw
intensity data into a. data file. In a particular embodiment,
modular software elements, such as for instance what may be
referred to as a plug-in module, may be interfaced with the core
software element to perform more specific or secondary functions,
such as for instance functions that are specific to particular
instruments. In particular, the specific or secondary functions may
include functions customizable for particular applications desired
by the user. Further, integrated modules and the core software
element are considered to be a single software application, and
referred to as applications.
[0047] Web Site Embodiment
[0048] A website or alternatively what is known in the art as
"cloud computing" embodiment may be used where typically; a user
(e.g., a scientist) executes a Web browser and is linked to a
server computer executing a Web server. In the present example some
commonly employed web browsers include Microsoft.RTM. Internet
Explorer 7 with SPI available from Microsoft Corporation, Mozilla
Firefox.RTM. 1.5 from the Mozilla Corporation, Safari 2.0 from
Apple Computer Corp., or other type of web browser currently known
in the art or to be developed in the future. The Web server may be
a program such as IBM's HTTP Daemon or other WWW daemon (e.g.,
LINUX-based forms of the program). The client computer may be
bi-directionally coupled with the server computer over a line or
via a wireless system. In turn, the server computer may be
bi-directionally coupled with a website (server hosting the
website) providing access to software implementing the methods of
this invention.
[0049] In particular embodiments, a user of a client connected to
the Intranet or Internet may cause the client to request resources
that are part of the web site(s) hosting the application(s)
providing an implementation of the methods or systems of this
invention. Server program(s) or APIs running on the server then
process the request to return the specified resources (assuming
they are currently available), e.g. a browser running an
application that displays the selected visualizations and metrics
of an implementation described above. A standard naming convention
has been adopted, known as a Uniform Resource Locator ("URL"). This
convention encompasses several types of location names, presently
including subclasses such as Hypertext Transport Protocol ("http"),
File Transport Protocol ("ftp"), gopher, and Wide Area Information
Service ("WAIS").
[0050] In a particular embodiment, when a resource is downloaded,
it may include the URLs of additional resources. Thus, the user of
the client may easily learn of the existence of new resources that
he or she had not specifically requested. One example of such an
implementation would be a user on a remote machine using a web
browser to make procedure calls, such as through an implementation
of XML-RPC, discussed at http://www.xmlrpc.com, or Simple Object
Access Protocol (SOAP), to a server application with an API for
implementing the system or methods of the invention. The user
through the web browser may be able to access all system
functionality through a remote location with a server running the
applications implementing the methods or systems of the
invention.
[0051] Methods of implementing Intranet and/or Intranet embodiments
of computational and/or data access processes are well known to
those of skill in the art and are documented, e.g., in ACM Press,
pp. 383-392; ISO-ANSI, Working Draft, "Information
Technology-Database Language SQL", Jim Melton, Editor,
International Organization for Standardization and American
National Standards Institute, July 1992; ISO Working Draft,
"Database Language SQL-Part 2: Foundation (SQL/Foundation)",
CD9075-2:199.chi.SQL, Sep. 11, 1997; and Cluer et al. (1992) A
General Framework for the Optimization of Object-Oriented Queries,
Proc SIGMOD International Conference on Management of Data, San
Diego, Calif., Jun. 2-5, 1992, SIGMOD Record, vol. 21, Issue 2,
Jun. 1992; Stonebraker, M., Editor. Other resources are available,
e.g., from Microsoft, IBM, Sun and other software development
companies.
[0052] Integrated Systems
[0053] Integrated systems, e.g., for performing flow cytometry
assays and data analysis, as well as for the compilation, storage
and access of databases, typically include a digital computer with
software including an instruction set as described herein, and,
optionally, one or more of high-throughput sample control software,
image analysis software, other data interpretation software, a
robotic control armature for transferring solutions from a source
to a destination (such as a detection device) operably linked to
the digital computer, an input device (e.g., a computer keyboard)
for entering subject data to the digital computer, or to control
analysis operations or high throughput sample transfer by the
robotic control armature. Optionally, the integrated system may
further comprise an image scanner for digitizing label signals from
labeled assay components.
[0054] In particular embodiments, readily available computational
hardware resources using standard operating systems may be employed
and modified according to the teachings provided herein, e.g., a PC
running for example, a Windows.RTM.-type operating system (such as
Windows.RTM. XP or Windows.RTM. Vista) from the Microsoft
Corporation; the Mac OS X operating system from Apple Computer
Corp. (such as 7.5 Mac OS X v10.4 "Tiger" or 7.6 Mac OS X v10.5
"Leopard" operating systems); a Unix.RTM. or Linux-type operating
system available from many vendors or what is referred to as an
open source; another or a future operating system; or some
combination thereof for use in the integrated systems of the
invention. Current art in software technology is adequate to allow
implementation of the methods taught herein on a computer system.
Thus, in particular embodiments, the present invention may comprise
a set of logic instructions (either software, or hardware encoded
instructions) for performing one or more of the methods as taught
herein. For example, software for providing the described data,
visualization, and/or statistical analysis may be constructed by
one of skill using a standard programming language such as Python,
Visual Basic, Fortran, Basic, Java, C++, C#, or the like. Such
software may also be constructed utilizing a variety of statistical
programming languages, toolkits, or libraries.
[0055] In particular embodiments, various programming methods and
algorithms, including genetic algorithms and neural networks, may
be used to perform aspects of the data collection, correlation, and
storage functions, as well as other desirable functions, as
described herein. In addition, digital or analog systems such as
digital or analog computer systems may control a variety of other
functions such as the display and/or control of input and output
files. Software for performing the visualization methods of the
invention, such as programmed embodiments of the visualization
methods described above, are also included in the computer systems
of the invention. Alternatively, programming elements for
performing such methods as principle component analysis (PCA) or
least squares analysis may also be included in the digital system
to identify relationships between data. Exemplary software for such
methods is provided by Partek, Inc., St. Peter, Mo.; available at
http://www.partek.com. Optionally, the integrated systems of the
invention include an automated workstation.
[0056] In particular embodiments, automated and/or semi-automated
methods for solid and liquid phase high-throughput sample
preparation and evaluation are available, and supported by
commercially available devices. For example, robotic devices for
preparation of nucleic acids from bacterial colonies, e.g., to
facilitate production and characterization of the libraries of
candidate genes include, for example, an automated colony picker
(e.g., the Q-bot, Genetix, U.K.) capable of identifying, sampling,
and inoculating up to 10,000/4 hrs different clones into 96 well
microtiter dishes. Alternatively, or in addition, robotic systems
for liquid handling are available from a variety of sources, e.g.,
automated workstations like the automated synthesis apparatus
developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and
many robotic systems utilizing robotic arms (Zymate II, Zymark
Corporation, Hopkinton, Mass.; Orca, Beckman Coulter, Inc.
(Fullerton, Calif.)) which mimic the manual operations performed by
a scientist. Additionally, incubators, refrigerators, freezers,
plate sealers, reagent dispensers, and barcode scanners may be
necessary to augment the robotic systems at the appropriate
processing steps. Any of the above devices are suitable for use
with the present invention, e.g., for high-throughput analysis of
library components or subject samples. The nature and
implementation of modifications to these devices (if any) so that
they may operate as discussed herein will be apparent to persons
skilled in the relevant art.
[0057] A variety of commercially available peripheral equipment,
including, e.g., flow cytometers and related optical and
fluorescent detectors, and the like, and software are available for
digitizing, storing and analyzing a digitized video or digitized
optical or other assay results using a computer. Commercial
Suppliers of flow cytometry instrumentation include Beckman
Coulter, Inc. (Fullerton, Calif.), and Becton Dickinson (San Jose,
Calif.), among many others.
[0058] In particular embodiments, applications may be capable of
data encryption/decryption functionality. For example, it may be
desirable to encrypt data, files, information associated with GUI's
or other information that may be transferred over network to one or
more remote computers or servers for data security and
confidentiality purposes. In some applications, the one-way
encrypted data may be stored in one or more public databases or
repositories where even the curator of the database or repository
would be unable to associate the data with the user or otherwise
decrypt the information. The described encryption functionality may
also have utility in clinical trial applications where it may be
desirable to isolate one or more data elements from each other for
the purpose of confidentiality and/or removal of experimental
biases.
[0059] Particular embodiments provide one or more interactive
graphical user interfaces that allow the user to make selections
based upon information presented in an embodiment of GUI. Those of
ordinary skill will recognize that embodiments of GUI may be coded
in various language formats such as an HTML, XHTML, XML,
javascript, Jscript, or other language known to those of ordinary
skill in the art used for the creation or enhancement of "Web
Pages" viewable and compatible with internet client, or other
languages known or developed in the future for use in the creation
of computer GUIs. For example, the internet client may include
various internet browsers such as Microsoft Internet Explorer,
Mozilla Firefox, Apple Safari, or other browsers known in the art.
Applications of GUI's viewable via one or more browsers may allow
the user complete remote access to data, management, and
registration functions without any other specialized software
elements. The applications may provide one or more implementations
of interactive GUI's that allow the user to select from a variety
of options including data selection, experiment parameters,
customization features, desired visualizations, metrics and
pre-filters.
[0060] In particular embodiments, the applications may be capable
of running on operating systems in a non-English format, where
applications may accept input from the user in various non-English
language formats such as Chinese, French, Spanish, etc., and output
information to the user in the same or other desired language
output. For example, applications may present information to the
user in various implementations of GUI in a language output desired
by the user, and similarly receive input from the user in the
desired language. In the present example, the applications are
internationalized such that it is capable of interpreting the input
from the user in the desired language where the input is acceptable
input with respect to the functions and capabilities of the
applications.
[0061] In particular embodiments, it may be desirable to
consolidate elements of data or metadata related to an experiment,
the user, or some combination thereof, to a single file that is not
duplicated where duplication may sometimes be a source of error.
The term "metadata" as used herein generally refers to data about
data. In particular embodiments, it may also be desirable in some
embodiments to restrict or prohibit the ability to overwrite data
in the file. Preferentially, new information may be appended to the
file rather than deleting or overwriting information, providing the
benefit of traceability and data integrity (e.g. as may be required
by some regulatory agencies).
[0062] The invention also may be embodied in whole or in part
within the circuitry of an application specific integrated circuit
(ASIC) or a programmable logic device (PLD). In such a case, the
invention may be embodied in a computer understandable descriptor
language, which may be used to create an ASIC, or PLD that operates
as herein described.
[0063] In a particular embodiment, a system to provide notice to
the operators of the cytometer lab that the samples are in transit
may be conducted over the internet. U.S. Patent Publication No.
20050009078 discusses the use of internet ordering systems useful
in the present invention.
[0064] Certain functional elements, such as files and data
structures, may be described in the illustrated embodiments as
located in system memory of a particular computer. In particular
embodiments, however, they may be located on, or distributed
across, computer systems or other platforms that are co-located
and/or remote from each other. For example, any one or more of data
files or data structures described as co-located on and "local" to
a server or other computer may be located in a computer system or
systems remote from the server.
[0065] In addition, it will be understood by those skilled in the
relevant art that control and data flows between and among
functional elements and various data structures may vary in many
ways from the control and data flows described above or in
documents incorporated by reference herein. More particularly, in
particular embodiments, intermediary functional elements may direct
control or data flows, and the functions of various elements may be
combined, divided, or otherwise rearranged to allow parallel
processing or for other reasons. In particular embodiments,
intermediate data structures or files may be used and various
described data structures or files may be combined or otherwise
arranged. Numerous other embodiments, and modifications thereof,
are contemplated as falling within the scope of the present
invention as defined by appended claims and equivalents
thereto.
[0066] Methods
[0067] Particular embodiments of the automation system perform
various methods of cell preparation for multiparametric analysis on
a flow cytometer. It is understood by the skilled artisan that the
steps of the methods provided herein may vary in order. It is also
understood, however, that while various options (of reagents,
handling properties selected, or order of steps) are provided
herein, the options are also each provided individually, and may
each be individually segregated from the other options provided
herein. Moreover, steps that are known in the art that may increase
the sensitivity of the assay are intended to be within the scope of
this invention. For example, there may be additional washing steps,
blocking steps, etc. It is understood by the skilled artisan that
steps of the methods provided herein may vary based on application,
such that some steps may be added or deleted depending on the
application.
[0068] FIG. 1 illustrates a high throughput system and method for
high throughput flow cytometry 100. In step 101, cell samples may
be prepared for processing. In step 103, cells are contacted with
one or more modulators in to stimulate the cells, then the cells
are fixed with a buffer containing reagent. Then the cells may be
permeabilized in step 105 to allow reagent access across the cell
membrane. In step 107, additional reagents are contacted with the
cells to "stain" particular proteins within the cell. Thereafter,
the cells are analyzed on a flow cytometer in step 109 to detect
the presence or absence of the stains. See U.S. Pat. Nos. 7,381,535
and 7,393,656 or application Ser. Nos. 10/193,462; 11/655,785;
11/655,789; 11/655,821; and 11/338,957 which are all incorporated
by reference in their entireties. See also 61/048,886; 61/048,920;
61/048,657; 61/079,766, and 61/085,789 which are also hereby
incorporated. As illustrated in FIG. 5-FIG. 9, high through system
and method for high throughput flow cytometry 100 may be utilized
with a variety of applications that all result in analysis of cells
on a flow cytometer, as illustrated in step 109.
[0069] The instant invention also makes use of cells that have been
"potentiated." In contrast to "activation," a "potentiated" state
refers the state of a cell after exposure to a modulator which then
may be activated as the case may be. As described in detail below,
modulators exert their effect on signaling cascades by directly or
indirectly impacting the ability of an activatible protein to
switch between activation isoforms.
[0070] Sample Acquisition
[0071] Many of the methods and applications described herein
require samples of cells on which to perform various analyses.
Cells may be acquired from variety of sources using a variety of
techniques. Regardless of the collection technique used, it is
important that there are a sufficient number of cells for the flow
analysis. In particular embodiments, cells may be acquired from an
individual, by a blood draw, a marrow draw, or a tissue extraction.
In particular embodiments, cells may be acquired by a variety of
other techniques and may include sources, such as bone marrow,
solid tumors, ascites, washes and the like. In a particular
embodiment, tissue may be taken from an individual using a surgical
procedure. Surgical procedures to acquire tissue are well known in
the art, as are subsequent processing of the tissue. Tissue may be
fixed or unfixed, fresh or frozen, whole or disaggregated.
Disaggregation of tissue may occur either mechanically or
enzymatically. In particular embodiments, cells may be cultured.
The cultured cells may be developed cell lines or patient derived
cell lines. Procedures for cell culture are commonly known in the
art.
[0072] In particular embodiments, once cell samples have been
collected they may be stored for later usage, processed and stored
for later usage, processed and used immediately, or simply used
immediately. In particular embodiments, processing may include
various methods of treatment, isolation, purification, filtration,
or concentration. Particular embodiments of high throughput flow
cytometry system and method may utilize fresh or cryopreserved
samples of blood, bone marrow, peripheral blood, tissue, or cell
cultures. Particular embodiments may utilized samples that are
activated or unactivated, fixed or unfixed, lyophilized or in
suspension. In a particular embodiment, processing may be performed
by an automated component, such as the micropipetting assembly
illustrated in FIG. 4. In a particular embodiment, an instrument
may access the processing method by a computer readable tangible
medium storing a computer program that contains processing
instructions for various types of cell samples.
[0073] In particular embodiments, preparation of the cell samples
may involve multiple steps. These steps may require multiple
reagents at various times in the process and it is preferable to
add the reagents soon after the blood drawing. Preferably, the
reagents are added within 0, 3, 5, 10, 15, 20, 30, 45, or 50
minutes or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 24, 30, or 36
hours after collection. In one embodiment, the cells are collected
directly by venous puncture and treated with the reagents. See U.S.
Patent Application No. 61/048,657.
[0074] In particular embodiments, blood may be treated without
processing (whole blood) or may be processed in some fashion. For
example, subsets of cells within the blood may be filtered and
removed for further use and analysis using standard collection or
filtering devices. In particular embodiments, samples may be whole
blood, cell suspensions, cells in a buffy coat sample, or fixed to
a solid substrate, such as a bead or plate. Some cell concentration
devices or techniques may be employed by insertion between the
blood collection tube and the chambers. In a particular embodiment,
a filtering device that separates cells may be placed in a fluid
line between the blood collection tube and another component of the
system. Such devices may include micro card or lab-on-a-chip
devices that separate specific subsets of cells from the whole
blood. In a particular embodiment, whole blood may also be applied
to filters that are engineered to contain pore sizes that select
for the desired cell type or class. For example, cells may be
filtered out or diluted, whole blood following the lysis of red
blood cells by using filters with pore sizes between 5 to 10 .mu.m,
as disclosed in U.S. patent application Ser. No. 09/790,673. In a
particular embodiment, a larger volume of blood may be necessary
where the concentration of cells was low and filters may be used to
concentrate the cell sample. See U.S. patent application Ser. No.
09/790,630.
[0075] Alternately, after collection of the cells, particular
embodiments may place cell samples into chambers for processing
through a fluid connection between the tube and the chambers See
U.S. Patent Application No. 61/048,657. The fluid connection may be
any one of a manner of conduits (such as commercial lab tubing) of
any appropriate size and shape. Tubing is commercially available
through a variety of medical and research suppliers. See the
commercially available equipment from Corning, Becton-Dickinson,
Sastedt, and Tygon.
[0076] Sample Processing
[0077] High throughput flow cytometry system and method is designed
to facilitate automated and high throughput processing of cell
samples using phosphoflow cytometry techniques. High throughput
flow cytometry system may be a flexible instrument-based system
where one or more instruments may interact with each other.
Furthermore, particular embodiments may integrate improved
procedures to allow more samples to be analyzed in fewer steps or
to provide additional information to the user. Particular
embodiments may comprise several elements, such as one or more
instruments, stations, or computers. Particular embodiments may be
carried out by fewer elements or a single element. Additionally,
any or all of the steps outlined herein may be automated. In
particular embodiments, high throughput flow cytometry system may
be partially or completely automated.
[0078] FIG. 1 illustrates a particular embodiment of a system and
method for high throughput flow cytometry 100. In FIG. 1, system
and method for high throughput flow cytometry 100 comprises islands
of automation 101, 103, 105, 107, 109. In particular embodiments,
any functional element of FIG. 1 may perform fewer or different
operations than those described with respect to the illustrated
embodiment. Also, various functional elements shown as distinct for
purposes of illustration may be incorporated within other
functional elements in a particular embodiment. In particular
embodiments, the sequence of functions or portions of functions may
be altered.
[0079] FIG. 2 illustrates a particular embodiment of a system and
method for high throughput flow cytometry 200. In FIG. 2, system
and method for high throughput flow cytometry 200 comprises islands
of automation 201, 203, and 205. In particular embodiments, any
functional element of FIG. 2 may perform fewer or different
operations than those described with respect to the illustrated
embodiment. Also, various functional elements shown as distinct for
purposes of illustration may be incorporated within other
functional elements in a particular embodiment. In particular
embodiments, the sequence of functions or portions of functions may
be altered.
[0080] FIG. 3A and FIG. 3B illustrate particular embodiments of a
fully automated high throughput flow cytometry system 300 where the
instrumentation is within a single platform or footprint. As will
be appreciated by those in the art, there are a wide variety of
components which may be used in association with the system.
Components include, but are not limited to, fixed or mobile plate
or reagent stages 301 with microfluidic systems; plate handlers 303
for the positioning of microplates that may have automated lid or
cap handlers to remove and replace lids; one or more robotic arms
305 that may be centrally located, as illustrated in FIG. 3A, or
proximately located to other components, as illustrated in FIG. 3B;
flow cytometer 307; automated centrifuges 309; heated, cooled, or
ambient reagent racks or stacking towers 311. Particular
embodiments may have one or more of the components previously
mentioned and one or more computer systems operatively linked to
one or more of those components. Particular embodiments may reduce
in size over time as instrumentation decreases in size or systems
may be simplified as processes become more efficient. Particular
embodiments may include an automated approach to inventory
management in addition to pathway profiling starting from sample
acquisition and ending with a full signaling pathway work-up.
[0081] Fully robotic or microfluidic systems include automated
liquid-, particle-, cell- and organism-handling including high
throughput pipetting and dispensing to perform all steps of
screening applications. This includes liquid, particle, cell, and
organism manipulations such as aspirating, dispensing, mixing,
diluting, washing, accurate volumetric transfers; retrieving and
discarding pipet tips; and repetitive pipetting of identical
volumes for multiple deliveries from a single sample aspiration.
These manipulations are cross-contamination-free liquid, particle,
cell, and organism transfers. This system performs automated
replication of microplate samples to filters, membranes, and/or
daughter plates, high-density transfers, full-plate serial
dilutions, and has high-capacity operation. In particular
embodiments, robotic or microfluidic systems may manipulate any
number of different reagents, including modulators, buffers,
fixatives, stains, permeabilizing reagents, reagent cocktails,
samples, wash solutions, assay components such as label probes,
etc.
[0082] Particular embodiments of high throughput flow cytometry
system may include one or more liquid handling components. The
liquid handling systems themselves may include robotic systems
comprising micropipetting assemblies and any number of other
components, such as the components illustrated in FIG. 3A and FIG.
3B. Example instruments that may be useful include, but are not
limited to, the Sciclone series i1000 or ALH3000 and its
Autostacker, or the Zepher from Caliper, the Biomek series,
including the NX.sup.P, NX, FX and the FX.sup.P from Beckman
Coulter, including the Bar Code Reading ALP, Stacker Carousel plate
hotel, Cytomat conveyor ALP, Automated Tube Bar Code Reader (once
microplates are formatted), or Handheld Bar Code Reader, the
Velocity 11 (Agilent) series, including the BioCel, BenchCel,
Bravo, and Vprep, the Tecan EVO liquid handling robots, the
Platemate, Hyrdra, or Wellmate liquid handlers and dispensers from
Matrix, or the Hamilton Robotics liquid handling workstations such
as the STAR or STARlet Lines, or the HyperCyt Autosampler by
Intellicyt or other such autosamplers. All of the above instruments
are shown on the websites for the relevant companies.
[0083] Micropippetting assemblies are well known in the art. See
U.S. Pat. No. 6,374,683. FIG. 4 illustrates an instrument 400 with
a micropipetting assembly 401, a user interface 403, a mobile plate
or reagent stage 405. Instrument 400 may be a stand-alone
instrument or operably linked to a computer system that contains
software for instrument control and operation.
[0084] The micropipetting assembly 401 may utilize syringe,
positive displacement, air displacement, peristaltic, or other
dispensing technology. The micropipetting assembly 401 may use
fixed or disposable tips that may be washable in a wash station.
The micropipetting assembly 401 may be movable in at least two
dimensions so that reagents or samples may be transferred from a
container to a microplate well, from a microplate well to an
alternate container, or between microplate wells. Particular
embodiments of micropipetting assembly 401 may transfer liquid
reagents or samples to and from microplate wells and reagent
troughs. In a particular embodiment, micropipetting assembly may
comprise a plurality of independently controlled micropipetters. In
a particular embodiment, each micropipettor may be controlled
separately for aspiration and dispense volumes, for the precise
microplate wells that it will aspirate or dispense from, and for
timing of such aspiration and dispenses.
[0085] User interface 403 allows an operator to manually interact
with the instrument to create or execute a program to control the
operation of the instrument. Mobile plate or reagent stage 405
allows micropipetting assembly 401 to interface with one or more
plates or other vessels that may contain reagents or sample. In a
particular embodiment, mobile plate or reagent stage 405 may
include a modular platform that may accommodate heated, cooled, or
ambient deck positions, a variable speed orbital shaker, positions
for source and destination vessels, or sample and reagent
reservoirs, pipette tips, and an active wash station. In a
particular embodiment, mobile plate or reagent stage may be
accessible by other components, such as a plate handler like the
plate handler 305 illustrated in FIG. 3.
[0086] In particular embodiments, reagents may be delivered by a
dispenser capable of aliquotting fluids to individual wells along
an X-Y-Z axis. See U.S. Pat. No. 6,121,048. In particular
embodiments, the dispenser may utilize a piezoelectric pump,
pipettes, micropipettes, electrophoretic pumps, or mechanisms
adapted from ink-jet printing technology to dispense precise
amounts of fluids. In a particular embodiment, reagents may be
delivered to precise locations within a plate or cartridge. In a
particular embodiment, specified reactants may be delivered to
certain wells which may be identified by encoded information, such
as a bar code, RFID, or magnetic coding, controlled by a processor
such as a computer.
[0087] Particular embodiments of high throughput flow cytometry
system may include thermoregulating components. Thermoregulating
components may be used to stabilize the temperature of any number
of samples or reagents. Thermoregulating components may include,
but are not limited to, incubators, refrigerators, freezers, heat
exchangers, such as temperature controlled blocks, resistive
elements, thermoelectric modules, or conductive elements.
Thermoregulating components may regulate temperatures ranging from
-80.degree. C. to 100.degree. C. In particular embodiments, the
thermoregulating component may include a temperature detection
device, such as a thermocouple, which provides signals
corresponding to temperature readings to another component or a
computer. When a controller receives the signals corresponding to
the temperature readings it may adjust the power output in order to
maintain the selected temperature. In particular embodiments,
thermoregulating components may be placed in close proximity to the
other components for ease of access. In a particular embodiment,
robotic elements, such as robotic arm 305, illustrated in FIG. 3A
and FIG. 3B, may be used to transport microplates from the
thermoregulating components to any other component. In a system as
illustrated in FIG. 1, microplates may be transferred to and from
thermoregulating components by hand from one automated component to
the next.
[0088] Optionally, a temperature control mechanism such as a
heater, a cooler, or a combination thereof may be disposed next to
the cartridge. The temperature control mechanism may be any
suitable thermally controlled element such as. The temperature
control mechanism transfers heat or cold via conduction to the
cartridge, which transfers heat or cold to fluid in the chamber.
Alternatively, the temperature control mechanism sinks heat away
from, for example, fluid in the chamber. The temperature control
mechanism maintains a selected temperature in the chamber. The
temperature control mechanism also
[0089] Particular embodiments of high throughput flow cytometry
system may utilize microtiter type plates. The plate may be
conventional and commercially available, or it may be a custom
design. The number of wells may be 96, 384, 1536 or other standard
sizes. The volume may be as stated above, at least 1, 2, 3, 4, 5,
6, or 7 or more milliliters. Microtiter plates may be obtained from
commercial suppliers such as those listed above. In a particular
embodiment, the microtiter plate may have predeposited
reagents.
[0090] Particular embodiments of high throughput flow cytometry
system may include chemically derivatized particles, plates,
cartridges, tubes, magnetic particles, or other solid phase matrix
with specificity to the assay components are used. The binding
surfaces of microplates, tubes or any solid phase matrices may
include non-polar surfaces, highly polar surfaces, modified dextran
coating to promote covalent binding, antibody coating, affinity
media to bind fusion proteins or peptides, surface-fixed proteins
such as recombinant protein A or G, nucleotide resins or coatings,
and other affinity matrix are useful in this invention.
[0091] In a particular embodiment, plates or tubes may have
preloaded reagents. In a particular embodiment, the plates may have
separate wells or compartments, and each well or compartment may be
capable of containing a separate reagent. These reagents may be
shown in the pending U.S. applications referred to above, such as
U.S. Patent Application No. 61/048,657 or 61/108,803.
[0092] Particular embodiments of high throughput flow cytometry
system may include platforms for multi-well plates, multi-tubes,
holders, cartridges, minitubes, deep-well plates, microfuge tubes,
cryovials, square well plates, filters, chips, beads, and other
solid-phase matrices or platforms with various volumes are
accommodated on an upgradable modular platform for additional
processing. Particular embodiments may include a variable speed
orbital shaker, and multi-position work decks for source samples,
source and destination assay plates, sample and reagent reservoirs,
pipette tips, and an active wash station.
[0093] Particular embodiments of the system may use components of
an automated handling apparatus similar to the simplified schematic
perspective drawing in FIG. 7 of U.S. Pat. No. 7,314,595. Example
components include a microplate carousel optionally situated within
environmental control chamber. In some embodiments, one or more
environmental control chambers may also be provided for other
components. Temperatures for the chambers may range from a low of
-80.degree. C., -20.degree. C., or 4.degree. C. to higher
temperatures of 30.degree. C., 60.degree. C., 95.degree. C. or
100.degree. C. In particular embodiments, the carousel and chamber
may be structurally connected to other components by a frame. In a
particular embodiment, a microplate robot may selectively move a
microplate stored in slots of carousel to a microplate retainer by
employing aspects of sensors and motors/actuators under control of
executables. In a particular embodiment, an operator may manually
place or remove the microplates from the chambers.
[0094] Particular embodiments of the system may include components,
such as the components illustrated in FIGS. 11-12 of U.S. Pat. No.
7,314,595. For example, FIG. 11 illustrates a robot that contains
mechanisms for holding a microplate, removing its lid, and
identifying the microplate by reading a one- or two-dimensional
barcode or employing any of various other machine-readable
indicators (e.g., signal transmitter or transducer, or other
device, any one or more of which are referred to for convenience
herein simply with reference to "barcode") known to those of
ordinary skill in the relevant art. In a particular embodiment the
robot may also return a microplate to the carousel after it has
been used, although microplates that have been processed may also
be stored elsewhere. In a particular embodiment, the robot may not
need to remove the lid prior to scanning the barcode or it may
return it to an alternate position within the instrument other than
the carousel or plate stacker. In a particular embodiment, an
operator may manually scan the microplate or barcodes using a
handheld barcode scanner.
[0095] Particular embodiments of the system may include microplate
retainers, such as those illustrated in FIGS. 15A-C and 16A-C of
U.S. Pat. No. 7,314,595. In a particular embodiment, the microplate
retainer may utilize fiducial features for registering bottom
surfaces of the microplates to a retainer. (See also aspects of the
use of reference marks and other features and techniques as
described in U.S. Pat. No. 6,121,048, hereby incorporated herein by
reference in its entirety for all purposes.)
[0096] In particular embodiments, once a microplate is secured in a
retainer, sampling equipment may be lowered into selected or
predetermined microplate wells to add or remove reagents, cells or
other reactants. In particular embodiments, the equipment may be
entirely submerged in the fluid material in the microplate well. In
particular embodiments, the equipment may either be kept stationery
while the microplate is moved to the equipment or move to where the
microplate is situated. The fluid volume in the equipment may be
0.001, 0.005, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
microliters, but usually not more than 10, 7.5, 5.0, 2.5 or 1.0
milliliters.
[0097] In particular embodiments of the system, flexible hardware
and software may allow instruments to be adaptable for multiple
applications. In a particular embodiment, the software program
modules may allow creation, modification, and running of one or
more methods. In a particular embodiment, the system diagnostic
modules may allow for instrument alignment, correct connections,
and motor operations. Additionally, customized tools, labware, and
liquid, particle, cell and organism transfer patterns may allow
different applications to be performed. In a particular embodiment,
the database may allow method and parameter storage, as well as
sample tracking or additional system controls. In a particular
embodiment, robotic and computer interfaces may allow communication
between one or more instruments and LIMS or LIS. U.S. Patent
Application Nos. 61/079,551 and 61/153,627 illustrate example
interfaces that are capable of storing experimental designs, plate
layout, and inventory management, and are hereby incorporated by
reference in their entirety. In a particular embodiment, the method
may be implemented through one or more instruments accessed by a
computer readable tangible medium storing a computer program
implementing the method's functionality.
[0098] Particular embodiments of high throughput flow cytometry
system and method may include a system for automated processing in
small volumes using a sample processing cartridge. Typical reaction
volumes in a sample processing cartridge may be 1 .mu.l to 100 ml.
Particular embodiments are capable of distributing specified and
accurate liquid volumes to the selected chambers. Appropriate
electronic and manual valve devices may be used to ensure that
liquid is placed in the correct chambers. For example, the
micropipetting assemblies recited above may be used to measure
liquid volumes. Valves may include standard physical devices that
are present on laboratory equipment that rotate with manual
stopcocks or electronic valves, or may include the types of valves
or liquid shunting devices or methods that are present with
lab-on-a-chip type devices where liquid volumes may be transferred
to selected chambers using positive or negative pressures or other
valving. See for example U.S. Pat. No. 6,830,936 and U.S.
Publication Nos. 2007157973; 20060258019; 20020079008; 20060134599;
and 20020125139. See also Seiler, K. et al., "Electroosmotic
Pumping and Valveless Control of Fluid Flow Within a Manifold of
Capillaries on a Glass Chip," Anal. Chem. 66:3485-3491 (1994);
Cheng X, Irimia, et. al., Practical label-free CD4+ T cell counting
of HIV-infected subjects: A Microchip approach. Lab on a Chip 2007;
10:1039; Demirci U, Toner M., Direct etch method for microfluidic
channel and nanoheight post-fabrication by picoliter droplets,
Applied Physics Letters 2006; 88 (5), 053117; and Irimia D, Geba D,
Toner M., Universal microfluidic gradient generator, Analytical
Chemistry 2006; 78: 3472-3477.
[0099] In particular embodiments, if the cells of interest were
concentrated, then smaller volume cartridges may be used, such as
the lab-on-a-chip devices, referred to above. For cartridges that
are used in these devices a typical cartridge may be about 0.5 to
3'' wide, 0.5 to 4'' long, and 0.1 to 1'' high or more preferably 1
to 2'' wide, 1 to 3'' long, and 0.5 to 1'' high. Preferably, the
cartridge is of sufficient size to accommodate identification
labels, RFIDs, or bar codes in addition to the chambers. Cartridges
in which the chambers have a volume of between 2 and 10 mls, may be
between 2 to 10'' wide, 2 to 12'' long, and 1 to 3'' high, or more
preferably 5 and 9'' wide, 3 and 9'' long, and 1 to 2'' high.
[0100] In particular embodiments, the chamber of a cartridge may
have any conceivable size, shape, or orientation. Preferably, the
chamber has a volume sufficient to allow the cells to contact one
or more reagents, such as a modulator, and then be mixed with other
reagents, like a fixative, permeabilizing reagent or a stain. In a
particular embodiment, the chamber may be at least 0.2'' wide,
0.2'' long, and 0.2'' deep. For small volume chambers the volume
may be in the microliter scale, such as the volumes described
above.
[0101] In particular embodiments, the cartridge may be open on two
or more ends or access points. In a particular embodiment, the cell
sample may be inserted through an inlet, port or opening. In a
particular embodiment, there may be only one opening to insert the
cell sample if the appropriate reagents are pre-deposited in the
chambers awaiting cells. In a particular embodiment, the cartridge
may be constructed to be able to open it to insert the sample and
then to close it to incubate or otherwise mix the sample plus the
reagents. In a particular embodiment, the cartridge may have a
separate access point to add the reagents to the cell sample after
the sample has been inserted into the chamber. The reagents may be
added individually or in one operation.
[0102] In particular embodiments, a separate device, such as an
automated dispenser may add reagents to the cartridge. In
particular embodiments, the automated dispenser may be attached to
the cartridge through a cassette. The use of a cassette having
areas that align to the chambers of the cartridge may further
enable high-throughput processing of the samples in lieu of the
slower process of individual addition. In particular embodiments,
alignment marks and structures may be added to facilitate an
appropriate match between the cartridge and devices with the
reagents. Alternatively, the reagents may be added to the chambers
in the cartridge through the manifold opening once the manifold is
removed.
[0103] In particular embodiments, selected fluids may be introduced
into and out of the chamber via inlets. In particular embodiments,
the inlets are located at opposite ends of the chamber. Having
inlets located at opposite ends of the chamber may improve fluid
circulation and regulation of bubble formation in the chamber for
mixing. In a particular embodiment, the bubbles may be used to
agitate the fluid, thus increasing the contact between the one or
more modulators and cell samples. Other methods to mix the sample
are known in the art. In a particular embodiment, inlets are
located at the top and bottom end of the chamber at opposite
corners. Locating the inlet at the highest and lowest positions in
the chamber may facilitate the removal of bubbles from the chamber
if desired. In particular embodiments, internal structure may also
be used in the chamber to facilitate mixing. Such structure may be
pegs, posts, or other physical structure that may disrupt the fluid
flow to enhance mixing.
[0104] In particular embodiments, the cartridge may contain
chambers having an inlet for the cell sample and a separate inlet
for the reagents. In a particular embodiment, the reagents may be
added by attaching a structure with or without its own chamber,
which may align the reagents with the chambers for contact with the
cells. In a particular embodiment, the structure may also have a
connection to add further reagents according to a particular timed
schedule as shown in the patent applications identified above.
[0105] In particular embodiments, once the first reagent is added,
the cells and the fluid in the chamber may be subject to agitation
to improve contact of the all of the reagents with the cells. See
U.S. Pat. No. 6,399,365 for examples of agitation systems. The
agitation may involve external shaking or internal fluid
circulation. Ports or inlet may be used to add further reagents. In
particular embodiments, during any of these procedures, the
manifold may be removed and the chamber openings may be covered
with a seal to prevent leakage and evaporation. The cover (or any
other seal) may be attached via clips, clamps, screws, adhesives,
and other fasteners.
[0106] In particular embodiments, the cartridge may include a seal
at any opening of the chamber. In a particular embodiment, the seal
may be a septum composed of rubber, teflon/rubber laminate, or
other sealing material. The septum may be of the type commonly used
to seal and reseal vessels when a needle is inserted into the
septum for addition/removal of fluids. The septums, when seated in
the depressions, extend slightly above surface, which in some
embodiments is about 0.01'' to 0.05''.
[0107] It should be noted that the even distribution of fluid flow
through the chamber prevents dead zones from occurring in the
chamber. For example, the even distribution of fluid through the
chamber substantially prevents fluid from becoming substantially
quiescent at certain locations. It should also be noted that any of
the components mentioned above may be used in conjunction with the
cartridge.
[0108] Timing
[0109] In particular embodiments, the system may control the
timings for sample handling, reagent addition, and incubation. For
any given sample being processed, there may be appropriate timings
for sample handling, reagent addition, and incubation, which may
ensure data integrity and ease data analysis. In particular
embodiments, timings for sample handling, reagent addition, and
incubation may either be a very specific set time, for example, a
fixative may need to incubate for exactly 10 minutes, or a range of
acceptable times, for example, a stain may incubate a few hours to
overnight, producing acceptable results. In a particular
embodiment, the system may treat each sample the same in terms of
timings at each step of the automated process for sample handling,
reagent addition, and incubation.
[0110] Particular embodiments of the system may control timing in a
variety of ways. In a particular embodiment, where the system is
fully automated, the timing may be controlled by software,
instruments, or other auxiliary systems. In a particular
embodiment, the system may assure proper timing of stimulation,
incubation, and fixation by using a liquid handling instrument to
dispense a modulator and then a fixative into a sample while
controlling the timing of all other processes as well, such as
movement of the sample to shakers, incubators, hotels, or alternate
positions. In a particular embodiment, where the system is not
fully automated, the system may utilize human intervention, such as
operator-implemented controls or batch processing to regulate the
timing. In a particular embodiment of the system, each batch will
proceed through the process in the same controlled manner such that
each batch will be treated the same at each step of the sample
preparation process within the system. The batch size may be
determined by instrument limitations, limitations on sample
handling, reagent addition, or incubation times, as well as overall
processing time per sample or any number of other limitations
imposed by the process itself to maintain data integrity.
[0111] Particular embodiments of the system may incorporate an
additional method of handling appropriate timing with respect to
reagent addition, such as a mechanism for timed release of
reagents. Methods for timed release are known in the art and
include physical structure or chemical formulations that are
designed to release a reagent at a particular time after an event
like adding the sample. In a particular embodiment, physical
devices may include an enclosure like an ampoule that may be broken
to release a reagent. In a particular embodiment, other physical
devices may include structure having a porous nature that will
release a reagent over time, such as a porous membrane. Examples of
chemical formulations that allow the timed release of reagents
include, but are not limited to micelles, liposomes, cleavable
linkers attached to the reagent and/or another molecule, and the
like. See U.S. Pat. Nos. 6,004,572, 5,079,005, and 5,023,080.
[0112] Particular embodiments of the system may use cartridges to
prepare the samples. In a particular embodiment, an instrument to
process the samples in the cartridge may control the timings for
sample handling, reagent addition and incubation. In a particular
embodiment, the reagents may be added to the cartridge chambers
prior to the addition of the cell sample. This embodiment allows
the cartridge to be preloaded and may make the process simpler for
the user while still ensuring processing uniformity. In a
particular embodiment, the reagents may be added after the cell
samples are added to the cartridge chambers. This embodiment may
require manual addition of reagents and some local processing by
the operator or an instrument or mechanism that would automatically
add the reagents to all chambers accurately and efficiently.
Particular embodiments of the system may leave the cartridge
attached or remove the cartridge and seal it from the
environment.
[0113] System Controls
[0114] System controls are commonly used in the art to ease
troubleshooting, maintain or improve throughput, and ensure end
result data integrity. Particular embodiments of the automation
system may have controls at various points within the system.
Various system controls may be environmental controls, operator
controls, reagent controls, instrument controls, process quality
control checkpoints, and protocol controls.
[0115] In a particular embodiment, a system control for managing
and monitoring the automation system may itself be a system for
experimental design, layout, and inventory management. See U.S.
Patent Application No. 61/079,551, which is hereby fully
incorporated by reference in its entirety.
[0116] Particular embodiments of the automation system may have
system controls that interface with the automation system by way of
a laboratory integrated management system (LIMS) or a laboratory
information system (LIS). Particular embodiments of a LIMS or LIS
may be associated with a computer workstation that may contain
software applications, GUI, or instruments, and may automate sample
tracking from wherever the sample is initially input into the
automation system through to data analysis and completion. LIMS or
LIS may automate sample logging at particular process steps by
communicating with one or more applications or one or more
instruments. LIMS or LIS may log one or more additional information
related to the plate or sample, such as a scientist or an operator,
a reagent, an instrument, and a date or date and time of processing
the plate or sample. A particular embodiment of LIMS or LIS may be
integrated into a fully automated system. A particular embodiment
of LIMS or LIS may require manual logging of one or more samples,
one or more plates, one or more operators, one or more reagents,
one or more instruments, one or more processing date or times or
where samples enter or exit specified processes.
[0117] In a particular embodiment, a system control may be the
automatic titration to determine the optimal amount of binding
element. In another embodiment, a system control is the use of
"sample barcoding," discussed below, to quickly and efficiently
determine the optimal binding element concentration, thus reducing
overall stain consumption.
[0118] Reagents
[0119] As described in detail throughout, the invention provides
methods, compositions, devices and kits for high throughput flow
cytometry. In some embodiments, the invention provides methods,
compositions, devices and kits for phosphoflow cytometry and
receptor function analysis. As such, there are numerous reagents
commonly known in the art to be associated with various embodiments
of the invention. In particular embodiments, some reagents may be
used at multiple process steps throughout sample preparation while
others may be used only once. There are various reagents that may
be added to the samples, plates, or chambers before or after
addition of the cell sample, thus it is known in the art that the
order of reagent addition may vary. Reagents may come in a variety
of compositions, such as in solid (powder, lyophilized, etc.) or
liquid form and may be used in the process in these various forms
as well. Reagents may also be stored and used at various
temperatures throughout the process.
[0120] Modulators:
[0121] Modulators, also referred to as "stims," include chemical
and biological entities, and physical or environmental stimuli.
Modulators can act extracellularly or intracellularly. Chemical and
biological modulators may include growth factors, cytokines,
neurotransmitters, adhesion molecules, hormones, small molecules,
inorganic compounds, polynucleotides, antibodies, natural
compounds, lectins, lactones, chemotherapeutic agents, biological
response modifiers, carbohydrate, proteases and free radicals.
Physical and environmental stimuli may include electromagnetic or
particulate radiation, redox potential and pH, the presence or
absences of nutrients, changes in temperature, oxygen partial
pressure, ion concentrations and oxidative stress. Modulators may
be endogenous or exogenous and may produce different effects
depending on the concentration and duration of exposure to the
single cells or whether they are used in combination or
sequentially with other modulators.
[0122] In particular embodiments, the modulators may include
ligands for cell surface receptors (for example the modulators
include IL-2, EGF, GMCSF, etc). Examples of such receptor elements
include compounds or events that will activate hormone receptors,
cytokine receptors, steroid receptors, adhesion receptors and
growth factor receptors, including, but not limited to, PDGF-R
(platelet derived growth factor receptor), EGF-R (epidermal growth
factor receptor), VEGF-R (vascular endothelial growth factor), uPAR
(urokinase plasminogen activator receptor), ACHR (acetylcholine
receptor), IgE-R (immunoglobulin E receptor), estrogen receptor,
thyroid hormone receptor, integrin receptors (.beta.1, .beta.2,
.beta.3, .beta.4, .beta.5, .beta.6, .alpha.1, .alpha.2, .alpha.3,
.alpha.4, .alpha.5, .alpha.6), MAC-1 (.beta.2 and cd11b),
.alpha.V.beta.33, opioid receptors (mu and kappa), FC receptors,
serotonin receptors (5-HT, 5-HT6, 5-HT7), .beta.-adrenergic
receptors, insulin receptor, leptin receptor, TNF receptor
(tissue-necrosis factor), cytokine receptors (IL1-a, IL-b, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10. IL-12, IL-15,
IL-18, IL-21, CCR5, CCR7, CXCR4, CCR-1-10, CCL20), statin
receptors, FAS receptor, BAFF receptor, FLT3 receptor, GMCSF
receptor, and fibronectin receptor. Specifically contemplated are
modulators that specifically relate to any of the particular
receptors noted above, such as IL-2 for the IL-2 receptor, and
VEGF-R for the VEGF receptor, for example.
[0123] Commercially available modulators are: Phorbol 12-Myristate
13-Acetate (PMA); Thapsigargin, Lipopolysacharride (LPS), CD40L,
SCF, IGF-1, IL-6, IL-10, Etoposide, IL-3, SDF-1a/CXCL12,
HydroxyUrea, Z-VAD-FMK Caspase Inhibitor, G-CSF, Erythropoetin
(EPO), SDF-1B/CXCL12, IL-27, M-CSF, GM-CSF, FLT-3 Ligand, VEGF, and
TRAIL.
[0124] Other modulators may be selected from the group consisting
of H.sub.2O.sub.2, siRNA, miRNA, Cantharidin,
(-)-p-Bromotetramisole, Microcystin LR, Sodium Orthovanadate,
Sodium Pervanadate, Vanadyl sulfate, Sodium
oxodiperoxo(1,10-phenanthroline)vanadate,
bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm
olybdate, Sodium Tartrate, Imidazole, Sodium Fluoride,
.beta.-Glycerophosphate, Sodium Pyrophosphate Decahydrate,
Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV,
Cypermethrin, Dephostatin, Okadaic Acid, NIPP-1,
N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propion-
amide, .alpha.-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br,
.alpha.-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br,
.alpha.-Bromo-4-(carboxymethoxy)acetophenone,
4-(Carboxymethoxy)phenacyl Br, and
bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene,
phenyarsine oxide, Pyrrolidine Dithiocarbamate, and Aluminium
fluoride.
[0125] Inhibitors:
[0126] Inhibitors specifically prevent a specific type of cellular
activity. Phosphatase inhibitors prevent the phosphatase enzyme
from removing a phosphate group from a substrate, and thus
preventing a specific cellular activity. Phosphatase inhibitors may
be used to analyze potential response to treatment or analyze
non-response to treatment. Inhibitors may be used to correlate the
expression or transporter proteins with their functionality.
Inhibitors may be used to make correlations within subpopulations
of cells gated both for phenotypic markers denoting stages of
development along hematopoietic and lymphoid lineages, as well as
reagents that recognize the transporter proteins themselves.
[0127] Inhibitors may include, but are not limited to, 3 mM
hydrogen peroxide (H.sub.2O.sub.2), 3 mM H.sub.2O.sub.2+SCF and 3
mM H.sub.2O.sub.2+IFN.alpha., PSC833, cyclosporine, probenecid,
fumitremorgin C, Ko143, and reserpine.
[0128] Fixatives:
[0129] Particular embodiments of the system may utilize assay and
screening methods, such as include "fixing." Fixing may be
performed to preserve or "freeze" a cell in a certain state,
preferably so that an accurate representation of the structure of
the cell is maintained. In particular embodiments it may be
desirable to fix a cell to maintain the cell's original size and
shape, to minimize loss of cellular materials, and/or to retain the
reactivity and/or status of the cell's intracellular constituents
(for example, the cell's phosphorylation level).
[0130] In particular embodiments, cells may be fixed by any of a
variety of suitable chemical and physical methods. In a particular
embodiment, fixatives may be applied to the detection of both
surface and intracellular antigens. In a particular embodiment, a
fixing method may be compatible with multi-well plate format
assays. In a particular embodiment, one or more fixing agents may
be added to cells contained in the well of an assay plate. Cells
may be incubated in the presence of the fixing agent at a certain
temperature (for example at room temperature, i.e., between
18.degree. C. and 25.degree. C.) and for a certain period of time
(for example between 5 and 10 minutes). In a particular embodiment,
fixation of cells in whole blood preferably would hypotonically
lyse the red blood cells while simultaneously fixing and preserving
the white blood cells. An example whole blood fixative may be found
here:
http://www.bdbiosciences.com/external_files/pm/doc/tds/cell_bio/live/web_-
enabled/558049.pdf. In particular embodiments, excess fixing agent
may be removed after centrifugation by aspiration of the
supernatant.
[0131] Particular embodiments of the system may rely on
crosslinking and/or rapid dehydration agents, such as formaldehyde,
paraformaldehyde, glutaraldehyde, acetic acid, methanol, ethanol,
and acetone to fix the cells. In a particular embodiment, Ortho
PERIVIEAFIX.TM., or PERMIFLOW.TM. (1N VIRION, INC..TM. MI) may be
used as a fixative. In a particular embodiment, a fixative may
comprise 0.756%-0.85% formaldehyde, 25.4-30 mM DNBS, 6.9-6.92% DMSO
and 0.086-0.095% TWEEN.TM. 20 detergent, although many variations
may also be used.
[0132] In particular embodiments, wash buffers may be used to "fix"
a cell after stimulation with a modulator. Wash buffers are know in
the art. See for example, U.S. Pat. No. 7,326,577 and U.S.
Publication No. 2006/0141549, which are hereby incorporated by
reference in its entireties.
[0133] Particular embodiments may utilize fixatives or fixing
methods described in the following references: Francis C. Connelly
M C, Rapid single-step method for flow cytometric detection of
surface and intracellular antigens using whole blood, Cytometry.
1996 Sep. 1; 25(1):58-70; Jacobberger, J W, Flow Cytometric
Analysis of Intracellular Protein Epitopes. Immunophenotyping 2000;
361-409; S & Cheta N, Permeafix: a useful tool to detect
antigens and DNA in flow cytometry, Rom J Intern Med. 1997
January-December; 35(1-4):133-5; Pizzolo G, et al. Detection of
membrane and intracellular antigens by flow cytometry following
ORTHO PermeaFix fixation. Leukemia. 1994 April; 8(4):672-6;
Pizzolo, G, et al., Detection of membrane and intracellular
antigens by flow cytometry following ORTHO PermeaFix fixation.
Leukemia. 1994 April; 8(4):672-6); Metso, T, et al., Identification
of intracellular markers in induced sputum and bronchoalveolar
lavage samples in patients with respiratory disorders and healthy
persons. Respir Med. 2002 November; 96(11):918-26); U.S. Pat. No.
5,422,277 and U.S. Pat. No. 5,597,688.
[0134] Permeabilization:
[0135] Particular embodiments of the system may "permeabilize"
cells once the cells have been fixed. Permeabilization may be
performed to facilitate access to cellular cytoplasm or
intracellular molecules, components or structures of a cell. In
particular, permeabilization may allow an agent (such as a
phospho-selective antibody) to enter into a cell and reach a
concentration within the cell that is greater than that which would
normally penetrate into the cell in the absence of such
permeabilizing treatment. In particular embodiments, cells may be
stored following permeabilization or combined with labeled binding
elements and then analyzed. In a particular embodiment, cells may
be permeabilized in the presence of 90% methanol and incubated on
ice for 30 minutes. Following this treatment, the assay plate may
be stored at -20.degree. C. for prior to being analyzed.
[0136] In particular embodiments, permeabilization of the cells may
be performed by any suitable method. Selection of an appropriate
permeabilizing agent and optimization of the incubation conditions
and time may easily be performed by one of ordinary skill in the
art. See for example, C. A. Goncalves et al., Neurochem. Res. 2000,
25: 885-894. Suitable methods include, but are not limited to,
exposure to a detergent (such as CHAPS, cholic acid, deoxycholic
acid, digitonin, n-dodecyl-.beta.-D-maltoside, lauryl sulfate,
glycodeoxycholic acid, n-lauroylsarcosine, saponin, and triton
X-100) or to an organic alcohol (such as methanol and ethanol).
Other permeabilizing methods comprise the use of certain peptides
or toxins that render membranes permeable. See, for example, O.
Aguilera et al., FEBS Lett. 1999, 462: 273-277; and A. Bussing et
al., Cytometry, 1999, 37: 133-139. Permeabilization may also be
performed by addition of an organic alcohol to the cells.
[0137] Binding Element
[0138] In some embodiments of the invention, the activation level
of an activatable element is determined Thus, the methods,
compositions, devices and kits of the invention may be employed to
examine and profile the status of any activatable element in a
cellular pathway, or collections of such activatable elements.
Activatable elements include intracellular or extracellular
biomolecules such as proteins, RNA, DNA, carbohydrates,
metabolites, and the like. Activatable elements are described in
U.S. application Ser. No. 12/460,029 filed Jul. 10, 2009; Ser. No.
12/432,720 filed Apr. 29, 2009; and Ser. No. 12/229,476 filed Aug.
21, 2008, incorporated herein by reference in their entirety.
Single or multiple distinct pathways may be profiled (sequentially
or simultaneously), or subsets of activatable elements within a
single pathway or across multiple pathways may be examined (again,
sequentially or simultaneously). As will be appreciated by those in
the art, a wide variety of activation events can find use in the
present invention. In general, the basic requirement is that the
activation results in a change in the activatable protein that is
detectable by some indication (termed an "activation state
indicator"), preferably by altered binding of a labeled binding
element or by changes in detectable biological activities (e.g.,
the activated state has an enzymatic activity which can be measured
and compared to a lack of activity in the non-activated state).
However, in other instances an activatable element gets activated
by increase expression. Thus, in those instances the increase
expression of the activatable element will be measured whether or
not there is a moiety between two or more activation states of the
cells.
[0139] In general, there are a variety of ways to detect the
activation state of a particular protein (i.e. activatible
element). Particular embodiments of the system may utilize the
state of the activatable protein itself by directly assaying the
activity or lack of activity within the signaling domains. In this
embodiment, for example, the two isoforms may be no activity
(negative signal) versus kinase activity (measured using
chromogenic substrates).
[0140] In some embodiment, the activation level of an activatable
element is determined by contacting a cell with a binding element
("BE") that is specific for an activation state of the activatable
element. The term "Binding element" includes any molecule, e.g.,
peptide, nucleic acid, small organic molecule which is capable of
detecting an activation state of an activatable element over
another activation state of the activatable element. Binding
elements and labels for binding elements are shown in U.S. Ser. No.
/048,886; 61/048,920 and 61/048,657.
[0141] In some embodiments, the binding element is a peptide,
polypeptide, oligopeptide or a protein. The peptide, polypeptide,
oligopeptide or protein may be made up of naturally occurring amino
acids and peptide bonds, or synthetic peptidomimetic structures.
Thus "amino acid", or "peptide residue", as used herein include
both naturally occurring and synthetic amino acids. For example,
homo-phenylalanine, citrulline and noreleucine are considered amino
acids for the purposes of the invention. The side chains may be in
either the (R) or the (S) configuration. In some embodiments, the
amino acids are in the (S) or L-configuration. If non-naturally
occurring side chains are used, non-amino acid substituents may be
used, for example to prevent or retard in vivo degradation.
Proteins including non-naturally occurring amino acids may be
synthesized or in some cases, made recombinantly; see van Hest et
al., FEBS Lett 428:(1-2) 68-70 May 22, 1998 and Tang et al., Abstr.
Pap Am. Chem. 5218: U138 Part 2 Aug. 22, 1999, both of which are
expressly incorporated by reference herein.
[0142] Particular embodiments of the system may utilize labeled
binding elements, or stains, or secondary stain reagents, which
bind specifically to one isoform of the protein. In some
embodiments, the protein BE may be an antibody. By "antibody"
herein is meant a protein consisting of one or more polypeptides
substantially encoded by all or part of the recognized
immunoglobulin genes. The recognized immunoglobulin genes, for
example in humans, include the kappa (k), lambda (I), and heavy
chain genetic loci, which together comprise the myriad variable
region genes, and the constant region genes mu (u), delta (d),
gamma (g), sigma (e), and alpha (a) which encode the IgM, IgD, IgG,
IgE, and IgA isotypes respectively. Antibody herein is meant to
include full length antibodies and antibody fragments, and may
refer to a natural antibody from any organism, an engineered
antibody, or an antibody generated recombinantly for experimental,
therapeutic, or other purposes as further defined below. The term
"antibody" includes antibody fragments, as are known in the art,
such as Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding
subsequences of antibodies, either produced by the modification of
whole antibodies or those synthesized de novo using recombinant DNA
technologies. Particularly preferred are full length antibodies
that comprise Fc variants as described herein. The term "antibody"
comprises monoclonal and polyclonal antibodies. Antibodies can be
antagonists, agonists, neutralizing, inhibitory, or
stimulatory.
[0143] Many antibodies, many of which are commercially available
(for example, see Cell Signaling Technology's and Becton
Dickinson's catalogues, the contents which are incorporated herein
by reference) have been produced which specifically bind to the
phosphorylated isoform of a protein but do not specifically bind to
a non-phosphorylated isoform of a protein. Many such antibodies
have been produced for the study of signal transducing proteins
that are reversibly phosphorylated. In particular, many such
antibodies have been produced which specifically bind to
phosphorylated, activated isoforms of protein kinases and are
sometimes referred to herein as kinase activation state antibodies
or grammatical equivalents thereof. Particularly preferred
antibodies for use in the present invention include: phospho-AKT
Ser473 monoclonal anti-4E2, phospho-p44/42 MAP kinase
(Thr202/Tyr204) monoclonal antibody, phospho-TYK2 (Tyr1054/1055)
antibody, phospho-p38 MAP kinase (Thr180/Tyr182) monoclonal
antibody 28B10, phospho-PKC-PAN substrate antibody,
phospho-PKA-substrate, phospho-SAPK/JNK (Thr183/Tyr185) G9
monoclonal antibody, phospho-tyrosine monoclonal antibody
(P-tyr-100), p44/42 MAPK, p38 MAPK, JNK/SAPK, and
phospho-AKT-Thr308.
[0144] In a particular embodiment, the protein BE may be an
activation state-specific antibody. Accordingly, the automation
system may use the methods and compositions described to detect any
particular element isoform in a sample that is antigenically
detectable and antigenically distinguishable from other isoforms of
the activatible element. In a particular embodiment, the system may
use the activation state-specific antibodies to identify distinct
signaling cascades of a subset or subpopulation of complex cell
populations, and the ordering of protein activation (e.g., kinase
activation) in potential signaling hierarchies.
[0145] In a particular embodiment, the BE may be a stain, where
stain may also be referred to as a detectable element, label or a
tag. By label is meant a molecule that can be directly (i.e., a
primary label) or indirectly (i.e., a secondary label) detected. By
way of example, and not by way of limitation, a label may be
visualized and/or measured or otherwise identified so that its
presence or absence may be known. A compound may be directly or
indirectly conjugated to a label which provides a detectable
signal, e.g. radioisotopes, fluorescers, enzymes, antibodies,
particles such as magnetic particles, chemiluminescers, specific
binding molecules, or molecules that may be detected by mass
spectroscopy, etc. Specific binding molecules include pairs, such
as biotin and streptavidin, digoxin and antidigoxin, etc. In
particular example embodiments, labels may be optical fluorescent
and chromogenic dyes including labels, label enzymes radioisotopes,
and quantum dots.
[0146] Kits
[0147] Particular embodiments of the high throughput flow cytometry
system and method may utilize kits. Such kits may enable the
detection of activatable elements by sensitive cellular assay
methods, such as IHC and flow cytometry, which are suitable for the
clinical detection, prognosis, and screening of cells and tissue
from patients, such as leukemia patients, having a disease
involving altered pathway signaling. Particular embodiments may be
compatible with clinical or research applications, a highly
automated or less automated high throughput flow cytometry system,
and a wide variety of instruments. Particular embodiments may be
provided, marketed and/or promoted to health providers, including
physicians, nurses, pharmacists, formulary officials, and the like,
or be marketed directly to the consumer. Particular embodiments may
include reagents for sample preparation, consumable hardware, such
as plates or other vessels for carrying the reagents or providing a
reaction space or pipette tips or tubing, a computer readable
tangible medium storing a computer software program or software
files that contain kit details and processing methods required for
sample preparation, and information, such as package insert
materials or literature references. Particular embodiments may be
packaged in any suitable manner, but typically with all elements in
a single container.
[0148] In a particular embodiment, reagents for sample preparation
may include one or more of the state-specific binding elements
described herein, such as phospho-specific antibodies, and
modulators, fixatives, buffers, therapeutic agents, and the
like.
[0149] In a particular embodiment, one or more of the
state-specific binding elements may be phospho-specific antibodies
specific for the proteins of interest. Examples of antibodies that
may be used may be selected from the group: PI3-Kinase (p85, p110a,
p110b, p110d), Jak1, Jak2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav,
Tiam, Sos, Dbl, Nck, Gab, PRK, SHP1, and SHP2, SHIP1, SHIP2, sSHIP,
PTEN, Shc, Grb2, PDK1, SGK, Akt1, Akt2, Akt3, TSC1,2, Rheb, mTor,
4EBP-1, p70S6Kinase, S6, LKB-1, AMPK, PFK, Acetyl-CoAa Carboxylase,
DokS, Rafs, Mos, Tp12, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK1,4,
MLK3, ASK1, MKK4/7, SAPK/JNK1,2,3, p38s, Erk1/2, Syk, Btk, BLNK,
LAT, ZAP70, Lck, Cbl, SLP-76, PLC.gamma..quadrature., PL.gamma.2,
STAT1, STAT 3, STAT 4, STAT 5, STAT 6, FAK, p130CAS, PAKs, LIMK1/2,
Hsp90, Hsp70, Hsp27, SMADs, Rel-A (p65-NFKB), CREB, Histone H2B,
HATs, HDACs, PKR, Rb, Cyclin D, Cyclin E, Cyclin A, Cyclin B, P16,
p14Arf, p27KIP, p21CIP, Cdk4, Cdk6, Cdk7, Cdk1, Cdk2, Cdk9, Cdc25,
A/B/C, Abl, E2F, FADD, TRADD, TRAF2, RIP, Myd88, BAD, Bcl-2, Mcl-1,
Bcl-XL, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8,
Caspase 9, IAPs, Smac, Fodrin, Actin, Src, Lyn, Fyn, Lck, NIK,
I.beta..quadrature..quadrature.1.quadrature..quadrature.,
PKC.alpha., PKC.beta., PKC.gamma..quadrature..quadrature.P,
.beta.catenin, CrkL, GSK3.alpha., GSK3.beta., and FOXO.
[0150] In a particular embodiment, one or more of the
phospho-specific antibodies specific for the proteins selected may
be selected from the group consisting of Erk, Syk, Zap70, Lck, Btk,
BLNK, Cbl, PLC.gamma.2, Akt, RelA, p38, S6. In a particular
embodiment, one or more of the phospho-specific antibodies specific
for the proteins selected may be selected from the group consisting
of Akt1, Akt2, Akt3, SAPK/JNK1,2,3, p38s, Erk1/2, Syk, ZAP70, Btk,
BLNK, Lck, PLC.gamma., PLC.gamma.2, STAT1, STAT 3, STAT 4, STAT 5,
STAT 6, CREB, Lyn, p-S6, Cbl, NF-.kappa..quadrature.
[0151] In a particular embodiment, modulators may be included. In a
particular embodiment, one or more modulators may be selected from
the group consisting of H.sub.2O.sub.2, PMA, BAFF, April, SDF1
.alpha., CD40L, IGF-1, Imiquimod, polyCpG, IL-7, IL-6, IL-10,
IL-27, IL-4, IL-2, IL-3, thapsigardin and a combination
thereof.
[0152] In a particular embodiment, the state-specific binding
element may be conjugated to a solid support and to detectable
groups directly or indirectly. In a particular embodiment, the
reagents may also include ancillary agents such as buffering agents
and stabilizing agents, e.g., polysaccharides and the like. In a
particular embodiment, the reagents may also include other members
of the signal-producing system of which system the detectable group
is a member (e.g., enzyme substrates), agents for reducing
background interference in a test, control reagents, apparatus for
conducting a test, and the like. In a particular embodiment, one or
more therapeutic agents may be included. The kit may further
comprise a software package for data analysis of the physiological
status, which may include reference profiles for comparison with
the test profile.
[0153] In a particular embodiment, a computer readable tangible
medium, including discs, thumb drives, external hard drives,
contains a computer software program or software files that contain
kit details and processing methods required for sample preparation.
The software may include files for a specific reaction using
specific reagents. In a particular embodiment, the software may
include directions for carrying out the process of reagent
addition, incubation, fixing cells, and other processes described
herein. In a particular embodiment, the software program or
software files may govern the positioning of the instrument and the
amount of time the reagents are allowed to react with the cell
samples. In a particular embodiment, a computer readable tangible
medium is compatible with an instrument, such as the instrument
illustrated in FIG. 4.
[0154] Such kits may also comprise tools and reagents to isolate a
biological specimen from an individual. The kits of the invention
may also comprise tools and reagent to isolate one or more
components (e.g. cytokines) from the biological specimen.
[0155] Such kits may additionally comprise one or more therapeutic
agents. The kit may further comprise a software package for data
analysis of the physiological status, which may include reference
profiles for comparison with the test profile.
[0156] Particular embodiments may include information, such as
scientific literature references, package insert materials,
clinical trial results, and/or summaries of these and the like,
which indicate or establish the activities and/or advantages of the
composition, and/or which describe dosing, administration, side
effects, drug interactions, or other information useful to the
health care provider. Such information may be based on the results
of various studies, for example, studies using experimental animals
involving in vivo models and studies based on human clinical
trials.
[0157] "Sample Barcoding"
[0158] Particular embodiments of the automation system may be
adjusted to increase the throughput through the use of a process
referred to as "sample barcoding." In particular embodiments,
sample barcoding may reduce or eliminate well to well variation,
such as variations in staining and the resultant variation in
fluroescence. In particular embodiments, sample barcoding may
reduce overall antibody consumption. In particular embodiments,
sample barcoding may allow multiple samples that have been
processed differently to be combined into a single tube or well
prior to the staining step so that all samples may be analyzed
together in a single run on the flow cytometer. See Fluorescent
cell barcoding in flow cytometry allows high-throughput drug
screening and signaling profiling, Krutzik, P. and Nolan G., Nature
Methods, Vol. 3 No. 5, Pgs. 361-365, May 2006; see also U.S. Patent
Publication No. 20080241820, which are hereby incorporated by
reference in their entirety.
[0159] In particular embodiments, sample barcoding may occur during
permeabilization, where the samples are also labeled with the
fluorophore barcodes. In particular embodiments, samples may be
labeled with different intensities of fluorophores by treatments
with varying concentrations or combinations of the reactive forms
of the fluorophores, which may result in each sample having a
unique fluorescence intensity signature. In particular embodiments,
the use of multiple fluorophores may increase the number of
different samples that may be barcoded, combined into a single
well, and then analyzed together. In particular example
embodiments, one fluorophore may allow 4-6 samples to be analyzed
together, two fluorophores may allow 16-36 samples to be analyzed
together, and three fluorophores may allow 64-216 samples to be
analyzed together. The greater the number of flurophores used, the
greater the number of samples that may be analyzed together. In
particular embodiments, downstream analysis software may be used to
deconvolute the data from the single well back into separate
results for each sample using the fluorescent barcodes.
[0160] In particular embodiments, sample barcoding may replace
various techniques for optimizing the amount of binding element to
add to a particular sample. Binding element may be added to a
single sample well or tube containing both the unstim and the stim
samples, which may then be analyzed together. In particular
embodiments, false positives due to an overconcentration of
antibodies may be quickly and accurately identified in the unstim
cells by allowing the data from the unstim and stim to be
visualized together. In particular embodiments, sample barcoding
may indicate whether antibody concentrations are too weak or too
strong. In particular embodiments, sample barcoding may permit the
analysis of small shifts in cell populations that may not otherwise
be noticeable with other techniques.
[0161] In particular embodiments, sample barcoding may allow for
distinction between those cells that were dead at the beginning of
processing and cells that died as a result of processing. A cell
sample may be treated with one or more stains that are capable of
distinguishing between live and dead cells, such as an amine
reactive dye. Examples of stains include, but are not limited to,
Aqua, Alexa (Alexa 750 among other types of Alexa dyes), and Sytox
(available from Invitrogen or eBioscience). In a particular
embodiment, two or more stains may be used to detect the number of
cells that lost viability after various processing steps or at
various time intervals. In particular embodiments, sample barcoding
may allow gating between dead cells and live cells to remove dead
cells from further analysis, resulting in cells being divided into
three classifications.
[0162] Sample Analysis
[0163] Particular embodiments of the system may use a system or
method for high throughput sample analysis. In a particular
embodiment, an instrument may be employed that provides for fast
and automated sampling of microplate wells. Such an instrument may
be used to take the samples at the point that they are ready to be
analyzed by a flow cytometer or other detector. In a particular
embodiment, an instrument may deliver samples to the flow cytometer
for analysis.
[0164] An example instrument that is commercially available and may
be integrated into the system is the Hypercyt (Intellicyt,
Albuquerque, N. Mex.). The HyperCyt.RTM. high-throughput flow
cytometry approach is to fill the sample uptake/transport line with
a stream of discrete sample particle suspensions separated by an
air gap, each sample suspension being approximately 2 .mu.l or
larger if necessary. Particular embodiments of the system may use
1, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more microliters per sample. The
entire sample stream is continuously delivered to the flow
cytometer so that data from all the samples in a plate are acquired
and stored in a single data file. A high resolution time parameter
is also recorded during data acquisition. Temporal gaps in particle
detection are created in the data stream by the passage of the air
bubbles, allowing the individual particle suspensions to be
distinguished and separately evaluated when plotted in conjunction
with the time parameter. See U.S. Pat. No. 7,368,084;
http://www.intellicyt.com, and
http://www.intellicyt.com/download/SBS2007HyperCyt.pdf. In a
particular embodiment, the Hypercyt instrument may be operably
connected to the flow cytometer and provide a liquid connection for
samples to be inserted into the cytometer at a high rate.
[0165] Data Analysis
[0166] Automated sample processing for flow cytometry analysis
generates large amounts of data in various formats. Particular
embodiments of the automation system contain a system for automated
data analysis. In a particular embodiment, automated gating of
sample data may be used to automate data analysis. U.S. Patent
Application No. 61/079,579 is hereby fully incorporated by
reference in its entirety. In a particular embodiment, automation
system may utilize a method and system for data extraction and
visualization of multiparametric data. U.S. Patent Application No.
61/079,537, hereby fully incorporated by reference in its
entirety.
[0167] Applications
[0168] Applications of high throughput flow cytometry system and
methods may include, but are not limited to, probing various
pathways by analyzing responses to a host of stimuli, assessing the
inhibition or enhancement of various signaling pathways by the
additions of small compounds from a small compound library,
assessing expression level and functionality of various drug
transporters, or probing the signaling differences among various
cancer cell lines (ie, NCI 60 cell lines). Examples of drug
transporters include, but are not limited to, glycoprotein (MDR1),
MDR-associated protein and breast cancer resistance protein. In
particular embodiments, automation may be used to screen the
supernatants from hybridomas in order to pick the best antibody
secreting cell clone. In particular embodiments, automation may be
used to screen for a neutralizing or agonistic antibody to a growth
factor receptor by looking for the supernatant that best augments
the signaling downstream of the growth factor receptor. In
particular embodiments, automation may be used to select the best
cell clone secreting any protein that has a biological effect that
could be measured by its downstream effect on signaling. In
particular embodiments, high throughput system may be used to
determine the expression and functionality of cell surface proteins
prior to treatment and in response to various treatments related to
cancer therapy. Particular embodiments may analyze multiple
characteristics of the cell in parallel and after contact with the
therapy compound.
[0169] In particular embodiments, the system may be used for
running experiments or portions of experiments including, but not
limited to, apoptosis assays, cell stimulation assays, and kinetic
timepoint assays. FIG. 5 illustrates an example cell sample
preparation method for preparing apoptosis, standard, and kinetic
timepoint plates. The following processing steps are illustrated in
FIG. 5:
[0170] 1. Use laboratory information system (LIS) or other
automation software to initiate Liquid Handler Program.
[0171] 2. System requests appropriate stock stim/modulator to be
loaded.
[0172] 3. System requests appropriate diluents, tips, tubes and
plates for loading.
[0173] 4. System loads and reads barcodes for samples,
stims/modulators, tips, tubes, plates, etc.
[0174] 5. System performs Stim/Modulator dilutions to create
working solutions (WS) in tubes.
[0175] 6. System aliquots WS into Master Stim Plates for 96 Head
Stamping (All 96 wells and/or Row/Column Format).
[0176] 7. System aliquots samples and controls to designated
layouts in Apoptosis, Standard and Kinetic Timepoint Plates.
[0177] 8. System places standard and kinetic timepoint plates on
heated shakers. [0178] a. Temperature is controlled to
.+-.0.5.degree. C. [0179] b. Setpoint may be adjusted and
controlled between 30.degree. C. and 50.degree. C. [0180] c.
Shaking/Vortex may be adjusted and controlled between 200 rpm to
1500 rpm with 3 mm amplitude
[0181] 9. System logs all barcodes, source positions, volume
pipetted and destination positions.
[0182] FIG. 6 illustrates an example apoptosis assay method. The
following processing steps are further illustrated in FIG. 6:
[0183] 1. Apoptosis working solutions are pipetted according to
designated format and number of samples to achieve final
stim/modulator concentrations in each well. This step may require
scheduling software to maintain acceptable time requirements.
[0184] 2. Plate is moved to 37.degree. C. Temperature Controlled
CO2 Incubator for specified incubation time.
[0185] 3. Plate is brought out of CO2 Incubator and Fix/Lyse
Reagent added with temperature and time control around
fixation.
[0186] 4. Plate is centrifuged, aspirated and cells dispersed by
vortex.
[0187] 5. Permeablization Reagent is added to plate and stored for
designated time at -80.degree. C.
[0188] 6. Plate is centrifuged, aspirated, cells dispersed by
vortex and wash buffer added.
[0189] 7. Plate is centrifuged, aspirated and cells dispersed by
vortex.
[0190] 8. System loads Apoptosis Stain Cocktails, pipettes to
designated wells, vortexes to mix, and incubates under time and
temperature control (4.degree. C. or 25.degree. C.).
[0191] 9. Wash buffer is added, the plate is centrifuged,
aspirated, cells dispersed by vortex and wash buffer added.
[0192] 10. Plate incubates for set time with wash buffer.
[0193] 11. Plate is centrifuged, aspirated and cells dispersed by
vortex.
[0194] 12. Secondary stain reagents are loaded and pipetted
according to layout and number of samples.
[0195] 13. Plate is incubated for set time, wash buffer added, the
plate is centrifuged, aspirated, cells dispersed and resupsended to
a final volume and cell concentration.
[0196] 14. Samples are transferred from Deep Well to shallow well
plate for acquisition (or to tube based carousel for acquisition)
on cytometer.
[0197] FIG. 7 illustrates an example standard stimulation assay
method. FIG. 8 illustrates an example kinetic timepoint assay
method. Processing steps for a stimulation assay method and a
kinetic timepoint assay method may be similar, but may differ at
the step of adding the standardized modulator working solutions or
the kinetic timepoint modular working solutions respectively. The
following process steps illustrate example standard stimulation and
kinetic timepoint assay processing:
[0198] 1. Standard Stim Plate or Kinetic Timepoint Stim Plate is
moved to deep well heater/shaker and gently vortexed for
predetermined time to obtain desired stimulation temperature.
[0199] 2. Standardized Stim/Modulator working solutions and Kinetic
Timepoint Stim/Modulator working solutions are pipetted into plate:
[0200] a. For Standarized Stim/Modulator (as illustrated in FIG.
7): working solutions are already in desired Master Plate layout.
[0201] i. Working solutions are pipetted by 96 Head to all wells at
once. [0202] ii. Plate is maintained under Temperature Control on
the heater/shaker for specified stim incubation time. [0203] b. For
Kinetic Timepoint Stim/Modulator (as illustrated in FIG. 8):
working solutions are already in desired Master Plate layout.
[0204] i. At t=0, the first set of wells (rows or columns) have
stim/modulator added, timer is started and the plate is vortexed
briefly to assure uniform mixing and maintained at under
Temperature Control on the heater/shaker for specified stim
incubation time. [0205] ii. At an appropriate time point, the next
series of stim/modulators are added, vortexed briefly to assure
uniform mixing and maintained at under temperature control on the
heater/shaker for specified stim incubation time. 1, 2, 3, or more
iterations of this step may occur.
[0206] 3. This step may require scheduling software to maintain
acceptable time requirements. At designed Stim Time (t.sub.stim),
Fix/Lyse Reagent is added while maintaining temperature and time
control around fixation.
[0207] 4. Plate is centrifuged, aspirated and cells dispersed by
vortex.
[0208] 5. Perm Reagent is added to plate and stored for designated
time at -80.degree. C.
[0209] 6. Plate is centrifuged, aspirated, cells dispersed by
vortex and wash buffer added.
[0210] 7. Plate is centrifuged, aspirated and cells dispersed by
vortex.
[0211] 8. System loads Standard Stain Cocktails, pipets to
designated destination according to layout and number of samples,
vortex to mix and incubates under time and temperature control
(4.degree. C. or 25.degree. C.).
[0212] 9. Wash buffer is added, the plate is centrifuged,
aspirated, cells dispersed by vortex and wash buffer added.
[0213] 10. Plate incubates for set time with wash buffer.
[0214] 11. Plate is centrifuged, aspirated and cells dispersed by
vortex.
[0215] 12. Secondary Stain Reagents are loaded and pipetted
according to layout and number of samples.
[0216] 13. Plate is incubated for set time, wash buffer added, the
plate is centrifuged, aspirated, cells dispersed and resupsended to
a final volume and cell concentration.
[0217] 14. Samples are transferred from Deep Well to shallow well
plate for acquisition (or to tube based carousel for acquisition)
on cytometer.
[0218] FIG. 9 illustrates an example high throughput multi-drug
resistant functional efflux assay method that may be used to
determine expression and functionality of specific cell surface
proteins prior to and in response to a given therapy. The following
steps illustrate the high throughput multi-drug resistant
functional efflux assay method of processing:
[0219] 1. Use laboratory information system (LIS) or other
automation software to initiate Liquid Handler Program.
[0220] 2. Cell sample plates are prepared according to cell type by
either thawing and resuspending, resuspending, or isolating and
resuspending. RPMI 10% FCS buffer and/or RPMI 10% FBS buffer may be
added to each sample as necessary.
[0221] 3. Inhibitor is prepared to a specific concentration and
stored at 37.degree. C. until use.
[0222] 4. Appropriate amount of inhibitor is added to sample plate,
mixed, and incubated for 15 minutes at 37.degree. C.
[0223] 5. Dye solution is prepared from concentrated dye and
DPBS/0.5% BSA.
[0224] 6. Plates are removed from incubator 30 seconds prior to
adding dye solution, and then appropriate amount of dye solution is
added to sample plate, mixed, and incubated for 45 minutes at
37.degree. C.
[0225] 7. Plate is centrifuged, aspirated and cells dispersed by
vortex.
[0226] 8. Efflux buffer is prepared using RPMI 10% FCS.
[0227] 9. Appropriate amount of efflux buffer is added to sample
plate, incubated at 37.degree. C. for 20 minutes.
[0228] 10. Plate is centrifuged, aspirated and cells dispersed by
vortex.
[0229] 11. IgG staining solution is prepared and added to sample
plate, mixed, vortexed, and incubated on ice (or equivalent
temperature) for 5 minutes.
[0230] 12. Plate is centrifuged, aspirated and cells dispersed by
vortex.
[0231] 13. Specific antibody and isotype staining mixtures are
prepared and added to sample plate, mixed, vortexed, and incubated
on ice (or equivalent temperature) in the dark for 30 minutes.
[0232] 14. Plate is centrifuged, aspirated and cells dispersed by
vortex.
[0233] 15. Wash buffer is added, plate is centrifuged, aspirated,
cells dispersed by vortex.
[0234] 16. After final wash/aspiration, cells are dispersed and
resupsended to a final volume and cell concentration.
[0235] 17. Samples are transferred from Deep Well to shallow well
plate for acquisition (or to tube based carousel for acquisition)
on cytometer.
[0236] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. Many other schemes for distributing functions among the
various functional elements of the illustrated embodiment are
possible. The functions of any element may be carried out in
various ways in alternative embodiments. It should be understood
that various alternatives to the embodiments of the invention
described herein may be employed in practicing the invention.
Therefore, the present disclosure encompasses all changes,
substitutions, variations, alterations, and modifications to the
example embodiments herein that a person having ordinary skill in
the art would comprehend. Similarly, where appropriate, the
appended claims encompass all changes, substitutions, variations,
alterations, and modifications to the example embodiments herein
that a person having ordinary skill in the art would comprehend.
Thus, it is intended that the following claims define the scope of
the invention and that methods and structures within the scope of
these claims and their equivalents be covered thereby.
* * * * *
References