U.S. patent application number 13/257571 was filed with the patent office on 2012-05-24 for ensemble-decision aliquot ranking.
Invention is credited to Daniel T. Chiu, Jason S. Kuo, Perry G. Schiro.
Application Number | 20120129190 13/257571 |
Document ID | / |
Family ID | 42983110 |
Filed Date | 2012-05-24 |
United States Patent
Application |
20120129190 |
Kind Code |
A1 |
Chiu; Daniel T. ; et
al. |
May 24, 2012 |
ENSEMBLE-DECISION ALIQUOT RANKING
Abstract
Provided herein, among other aspects, are methods and
apparatuses for ranking aliquots from a suspension containing
bioparticles. In certain embodiments, the bioparticles may be
cells, organelles, proteins, DNAs, debris of biological origin,
microbeads coated with biological compounds, or viral particles. As
such, the methods and apparatuses provided herein may be used to
quantify rare cells such as circulating cancer cells, fetal cells
and other rare cells present in bodily fluids for disease
diagnosis, prognosis, or treatment.
Inventors: |
Chiu; Daniel T.; (Seattle,
WA) ; Schiro; Perry G.; (Seattle, WA) ; Kuo;
Jason S.; (Seattle, WA) |
Family ID: |
42983110 |
Appl. No.: |
13/257571 |
Filed: |
April 13, 2010 |
PCT Filed: |
April 13, 2010 |
PCT NO: |
PCT/US2010/030938 |
371 Date: |
February 1, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61168892 |
Apr 13, 2009 |
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Current U.S.
Class: |
435/7.23 ;
250/459.1; 435/287.2; 435/288.7; 435/34 |
Current CPC
Class: |
Y02A 50/30 20180101;
G01N 33/5302 20130101; G01N 33/6893 20130101; G01N 33/57407
20130101; Y02A 50/58 20180101; G01N 33/5308 20130101 |
Class at
Publication: |
435/7.23 ;
435/34; 435/288.7; 435/287.2; 250/459.1 |
International
Class: |
G01N 21/64 20060101
G01N021/64; C12M 1/34 20060101 C12M001/34; G01N 21/76 20060101
G01N021/76; C12Q 1/04 20060101 C12Q001/04 |
Claims
1. A method for detecting a rare particle in a fluid sample, the
method comprising the steps of: (a) detecting the presence or
absence of a rare particle in an aliquot of the fluid sample; (b)
assigning a value to the aliquot based on the presence or absence
of the rare particle; and (c) directing the flow or collection of
the aliquot based on the assigned value.
2. The method of claim 1, wherein the step of detecting the
presence of the rare particle comprises the sub-steps of: (i)
contacting the fluid sample with a detection reagent under
conditions suitable to transform the detection reagent into a
complex comprising said detection reagent and a rare particle; and
(ii) detecting the presence or absence of a complex formed in step
(i) in an aliquot of the fluid sample.
3. The method of claim 1, wherein the step of detecting the
presence of the rare particle comprises the sub-steps of: (i)
interrogating the aliquot with an external source of
electromagnetic radiation; and (ii) detecting fluorescence of the
rare particle.
4. The method of claim 1, wherein the step of detecting the
presence of a rare particle comprises detecting bioluminescence or
chemiluminescence of the rare particle.
5. The method of 1, wherein the rare particle is a rare cell.
6. The method of claim 1, wherein the aliquot contains more than
one rare particle.
7. The method of claim 1, wherein the fluid sample is a biological
fluid.
8. The method of claim 1, wherein the fluid sample is
simultaneously contacted with a plurality of differentiable
detection reagents each having a different specificity under
conditions sufficient to transform the plurality of detection
reagents into a plurality of complexes comprising the detection
reagents and a plurality of rare particles.
9. The method of claim 8, wherein the plurality of complexes are
detected simultaneously.
10. The method of claim 8, wherein the detection reagents are
differentiable by fluorescence at different wavelengths.
11. The method of claim 1, wherein the aliquot is assigned either a
first value if the aliquot contains a rare particle or a second
value if the aliquot does not contain a rare particle.
12. The method of claim 1, wherein said first value depends on
either the identity of the rare particle or the concentration of
the rare particle in the aliquot.
13. The method of claim 1, wherein multiple aliquots having the
same assigned value are pooled together.
14. The method of claim 1, wherein the detection step is performed
during continuous flow of the biological sample through a flow
channel.
15. The method of claim 1, wherein a plurality of aliquots of the
fluid sample are physically separated prior to the detection
step.
16. The method of claim 15, wherein the aliquots are partitioned
into separate flow channels or chambers prior to the detection
step.
17. A method for providing a subject a diagnosis or prognosis for a
condition associated with the presence of a rare particle in a
biological fluid, the method comprising the steps of: (a)
contacting a biological fluid from the subject with a detection
reagent under conditions suitable to transform the detection
reagent into a complex comprising said detection reagent and a rare
particle; (b) detecting the presence or absence of a complex formed
in step (a) in an aliquot of the biological fluid; (c) assigning a
value to the aliquot based on the presence or absence of a complex
formed in step (a); and (d) providing a diagnosis or prognosis to
the subject based on the assigned value.
18. An apparatus for detecting a rare particle in a fluid sample,
the device comprising: (a) at least a first input channel; (b) at
least two exit channels; (c) at least one detector capable of
detecting one or more rare particles in an aliquot of the fluid
sample; (d) a mechanism for directing the flow of the aliquot; and
(e) a computer capable of assigning a value to the aliquot based on
the presence, absence, identity, composition, or quantity of the
rare particles in the aliquot, wherein the computer is in
communication with the detector and the mechanism for directing the
flow of the aliquot.
19. The apparatus of claim 18, wherein said mechanism directs the
flow of the aliquot into either a first exit channel if the aliquot
contains a rare particle or a second exit channel if the aliquot
does not contain a rare particle.
20. The apparatus of claim 18, wherein said mechanism directs the
flow of an aliquot containing a rare particle into one of a
plurality of exit channels depending on the identity, composition,
or quantity of the rare particle.
21. The apparatus of claim 18, wherein the mechanism for directing
the flow of the aliquot comprises an electrode, a magnetic element,
an acoustic element, or an electro-actuated element.
22. The apparatus of claim 18, wherein the mechanism for directing
the flow of the aliquot comprises one or more electro-actuated
valves or pistons, wherein the valves or pistons control the flow
of a liquid in at least a first directional flow channel that
intersects with the first input channel and the two exit channels
at a first junction.
23. The apparatus of claim 18, wherein the detector is selected
from the group consisting of a camera, an electron multiplier, a
charge-coupled device (CCD) image sensor, a photomultiplier tube
(PMT), an avalanche photodiode (APD), a single-photon avalanche
diode (SPAD), and a complementary metal oxide semiconductor (CMOS)
image sensor.
24. The apparatus of claim 18, wherein the apparatus further
comprises an electromagnetic radiation source for interrogation of
the aliquot.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Provisional Application No. 61/168,892 filed Apr. 13, 2009,
expressly incorporated herein by reference in its entirety for all
purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
[0002] NOT APPLICABLE
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ON A COMPACT DISK
[0003] NOT APPLICABLE
BACKGROUND OF THE INVENTION
[0004] Body fluids are complex suspensions of biological particles
in liquid. Blood, for example, includes plasma and cells (red blood
cells, white blood cells, platelets) and the cells occupy about 55%
of blood. Plasma is mostly water and it transfers proteins, ions,
vitamins, enzymes, hormones, and other chemicals to cells in the
body.
[0005] Red blood cells are about 6 to 8 .mu.m in size and serve to
provide oxygen to cells. White blood cells are about 10 to 13 .mu.m
in diameter and they defend the body from disease as a part of an
immune system by fighting against foreign viruses and bacteria.
Platelets are the smallest cells, 1.5 to 3 .mu.m, and they stop
bleeding by forming blood clots.
[0006] Fluids in addition to blood, such as saliva, tear, urine,
cerebral spinal fluid as well as other body fluids in contact with
various organs (e.g. lung) contain mixtures of cells and
bioparticles. The type and amount of cells and bioparticles that
are present in a particular body fluid (e.g. blood) reveals
information about the health of the organism, and in the case of an
infected individual, information about the diagnosis and prognosis
of the disease.
[0007] Some cells or bioparticles are present in rare quantities
compared to the nominal concentrations of blood cells. Despite
their rare occurrence, these cells or bioparticles may be
intricately tied to significant events that take place in the body
that alter the health state of an individual. These cells are
commonly referred to as "rare cells".
[0008] For example, dissemination of cancer cells from the primary
tumor is an important factor governing the probability of relapse
and the survival rate in cancer patients. As cancer cells grow
unregulated and lose their ability to adhere to each other, they
can enter the blood and lymphatic circulation and circulate
throughout the body. These cells are commonly referred to as
Circulating Tumor Cells (CTC), Disseminated Tumor Cells (DTC),
Circulating Cancer Cells (CCC), Circulating Epithelial Cells (CEC),
Occult Tumor Cells (OTC), or other similar permutations to indicate
the mobile nature of these cells, in contrast to the specimens
obtained by direct biopsy of solid tumors. CTCs have been detected
in the blood of patients suffering from all major cancers:
prostate, ovarian, breast, gastric, colorectal, renal, lung,
pancreatic, and others.
[0009] In this fashion, tests that counts CTCs present in bodily
fluids have been developed to assist with providing a prognosis for
cancer patients. A "CTC test" can also be used to monitor a
patient's response to a particular treatment (e.g. radiation or
chemotherapy) protocol. Based on the results of CTC test, a cancer
patient may be able to avoid significant costs by minimizing
additional unnecessary and expensive diagnostic tests and
therapies, which are often times not covered by health insurance,
for example Computed Tomography (CT) or Positron Emission
Tomography (PET) scans, or shorten the drug treatments that are
ineffective.
[0010] However, CTCs are present in extremely low concentration in
the peripheral blood, estimated to be on the order of one tumor
cell per 10.sup.6 to 10.sup.7 mononuclear cells, which is
equivalent to one tumor cell per 0.5 ml to 5 ml of peripheral
blood. At such a low concentration, a sample with estimated 100
million mononuclear cells must be screened in order to detect at
least one CTC with 99.995% certainty. Using conventional
techniques, such as automatic digital microscopy (ADM) scanning at
a typical speed of 800 cells/second, would require 18 hours to
complete a sample that size, rendering it monetarily and temporally
impractical for clinical use.
[0011] For example, conventional flow cytometry may be employed to
determine the presence or quantity of CTCs in a blood sample.
However, flow cytometry requires that the cells are organized
linearly in a row and detect each cell singularly because
simultaneous detection of two cells cannot be interpreted correctly
using existing technology. In flow cytometry, laser beams are
focused such that they only illuminate a single particle at any
given time. For example, if one nonfluorescent cell traverses the
detection volume simultaneously with a fluorescent cell configured
to be detectable by the flow cytometer, the nonfluorescent cell,
being invisible to the flow cytometer, would be directed in the
same trajectory as the fluorescent cell. To avoid misinterpretation
in flow cytometry, cells suspensions are routinely either diluted
or slowed down to allow sufficient distance between the cells to
avoid overlapping of two cells within the detection volume. Current
state-of-the-art flow cytometers have an upper limit of sorting
100,000 objects/second.
[0012] As such, flow cytometry is not suitable for detecting or
recovering rare cells. Detecting cells one at a time (serially) and
making a decision on the trajectory of every cell is too
time-consuming when analyzing a large number of cells. For example,
since ten milliliter of blood contains approximately ten billion
cells, at 100,000 cells per second, which is the highest sorting
speed of state-of-the-art flow cytometer, it would take 100,000
second or 28 hours to completely sort the content of 10 mL. For
rare cell, often 7-15 mL of blood is required to collect a
statistically significant number of rare cells; using a flow
cytometer to recover rare cells is an impractical consumption of
clinical resources and can translate to a very high testing
cost.
[0013] As such, simple and cost/time-effective techniques are
needed for the detection and quantitation of rare particles and
cells in a fluid sample. The present invention satisfies these and
other needs by providing methods and apparatuses for the detection
of rare particles in fluid samples.
BRIEF SUMMARY OF THE INVENTION
[0014] Among other aspects, the present invention provides methods
and apparatuses that rapidly scan a large volume of a fluid for the
detection and or quantitation of desired bioparticles by ranking
aliquots. In one aspect, the concept employed, termed
Ensemble-Decision Aliquot Ranking ("eDAR"), is particularly useful
for detecting rare cells in biofluids.
[0015] In one aspect, the present invention provides a method for
detecting a rare particle in a fluid sample, the method comprising
the steps of detecting the presence or absence of the rare particle
in an aliquot of the fluid sample, assigning a value to the aliquot
based on the presence or absence of the rare particle, and
directing the flow or collection of the aliquot based on the
assigned value.
[0016] In a second embodiment, the present invention provides a
method for providing a subject a diagnosis or prognosis for a
condition associated with the presence of a rare particle in a
biological fluid, the method comprising the steps of detecting the
presence or absence of the rare particle in an aliquot of the
biological fluid, assigning a value to the aliquot based on the
presence or absence of the rare particle, and providing a diagnosis
or prognosis to the subject based on the assigned value.
[0017] In a third aspect, the present invention provides a device
for detecting a rare particle in a fluid sample, the device
comprising at least a first input channel, at least two exit
channels, at least one detector capable of detecting one or more
rare particles in an aliquot of the fluid sample, a mechanism for
directing the flow of the aliquot, and a computer capable of
assigning a value to the aliquot based on the presence, absence,
identity, composition, or quantity of the rare particles in the
aliquot, wherein the computer is in communication with the detector
and the mechanism for directing the flow of the aliquot.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 illustrates simultaneous detection of multiple
particles in an aliquot and the directions of aliquot.
[0019] FIG. 2 illustrates a mask with apertures to improve the
signal-to-noise ratio as used in eDAR.
[0020] FIG. 3 illustrates a mask with closely spaced apertures to
improve the signal-to-noise ratio as used in eDAR.
[0021] FIG. 4 illustrates an eDAR apparatus consisting of a single
flow channel, 2 lasers, and three detectors.
[0022] FIG. 5 illustrates an eDAR apparatus consisting of three
flow channels, a laser, and a detector.
[0023] FIG. 6 illustrates an eDAR apparatus consisting of three
flow channels, a laser, and three detectors.
[0024] FIG. 7 illustrates an eDAR apparatus consisting of five flow
channels, a laser, and three detectors.
[0025] FIG. 8 shows a comparison in circulating tumor cell (CTC)
counts from blood using eDAR (upper panel) and a commercial
instrument (Veridex's CellSearch, lower panel).
[0026] FIGS. 9A-C shows the optical images of cancer cells trapped
from patient blood using eDAR (arrows mark CTCs) under various
illumination. FIG. 9A is a brightfield image of a CTC amidst red
blood cells. FIG. 9B is a fluorescence image indicating the
presence of pan-cytokeratin. FIG. 9C is a fluorescence image
indicating the absence of CD45, hence ruling out the possibility of
false-identifying a white blood cell as a CTC.
[0027] FIGS. 10A-D show optical images of cancer stem cells
distinguished from ordinary cancer cells. Arrowheads indicate
cancer stem cells (CD44+/CD24-). FIG. 10A is a fluorescence image
(500-540 nm) for detecting Alexa 488-anti-CD44 (green); FIG. 10B is
a fluorescence image (645-700 nm) for detecting Alexa 647-anti-CD24
(red); FIG. 10C is the brightfield image. FIG. 10D is a composite
image indicating CD44+/CD24-(arrows indicate cancer stem
cells).
[0028] FIGS. 11A-D illustrate the operation of device 1110 for
aliquoting a suspension.
[0029] FIGS. 12A-C illustrates device 1210 used for aliquoting
suspension with five fluidic channels (1211, 1212, 1213, 1214, and
1215) joined at junction 1240.
[0030] FIG. 13 shows the fluorescence signals collected from 2
avalanche photodiodes (APDs) positioned at different locations
upstream and downstream of junction 1116 (or 1240). Plot 1310 shows
the signal trace 1311 from one APD configured to detect the
presence of EpCAM molecule in an aliquot at detection volume 1140
(or 1270), whereas Plot 1320 shows the signal trace 1321 from a
second APD configured to detect the presence of EpCAM molecule in
channel 1114 (or 1214).
[0031] FIG. 14 shows plot 1410 illustrating the percentage of
cancer cells recovered as a function of the pulse length using
eDAR. Trace 1420 indicates that when the pulse width was 10 ms or
below, 100% of the cancer cells were collected in the correct
channel.
[0032] FIG. 15 shows plot 1510 with trace 1520 indicating the
percentage of rare cells recovered as a function of incoming flow
rate.
[0033] FIG. 16 illustrates the use of discrete aqueous aliquots
separated by an immiscible phase to encapsulate bioparticles prior
to reaching the detection volume.
DETAILED DESCRIPTION OF THE INVENTION
[0034] I. Overview
[0035] In one aspect, the present invention provides methods and
apparatuses for detecting and/or recovering rare particles in a
fluid sample. The concept embodied in this aspect is referred to
herein as "Ensemble-Decision Aliquot Ranking" or "eDAR." In one
embodiment, the eDAR methodology can be characterized as (i)
detecting the presence of absence of a rare particle in an aliquot
of the fluid sample, (ii) ranking the aliquot according to the
presence or absence of a rare particle, and (iii) directing the
flow or collection of the entire aliquot based on the assigned
ranking.
[0036] For finding rare cells, eDAR offers a tremendous advantage
in speed. As an example of this, consider a 10-mL cell suspension
containing 10 desired rare cells. In this case, about 99.9999% of
the suspension volume does not contain a single desired rare cell.
Existing technologies, such as flow cytometry, FACS, etc.,
invariably scan serially through every cell contained within the
entire suspension volume, resulting in nearly all of time wasted
scanning through the 99.9999% of the suspension volume that does
not contain a desired cell. eDAR allows a quick high-level
screening of the entire suspension by aliquoting the suspension. If
the cells are indeed rare, most aliquots will not contain any
desired cells. In one embodiment, these aliquots are ranked as null
and discarded through a first channel. In comparison, the few
aliquots that do contain rare cells are ranked as nonzero and
collected in a separate channel or chamber.
[0037] In this fashion, eDAR is distinct from conventional flow
cytometry. Flow cytometry operates by (1) flowing bioparticles in
single file (i.e., one by one in a row) through the use of a sheath
flow to contain the particle-row, (2) detecting only one
bioparticle at a time, and (3) determine the trajectory of the
bioparticle detected. If the detected bioparticle is desired, for
example, the bioparticle is directed, through a one of a variety of
methods, to follow a certain trajectory to reach a collection
container. If the detected bioparticle is undesirable, the
bioparticle is directed in a different trajectory to reach a
different collection container.
[0038] In contrast to conventional flow cytometry, eDAR
interrogates entire aliquots, i.e., three-dimensional subdivisions
of a fluid, to make an ensemble decision for the entire aliquot.
Unlike serializing (1-D subdivision of fluid, which results in
bioparticles arranged in a single row) or planarizing (2-D
subdivision of fluid, which results in bioparticles arranged in
multiple parallel rows in a plane), aliquoting offers a much higher
throughput. Flow cytometry is incapable of analyzing an aliquot
because the detectors are configured only collect signals from a
single cell or a single plane of cells. Bioparticles outside of the
detection point or plane are entirely invisible to the detectors
used in flow cytometers and consequently sheath flow, guiding
buffers, or other hydrodynamic or geometric focusing mechanisms are
necessary to prevent migration of bioparticles outside of the
detection point or plane. eDAR analyzes an entire aliquot which
spans more than one plane or layer of bioparticles. In eDAR,
signals emanating from an entire aliquot, as opposed to a single
detection point or plane, are analyzed.
[0039] eDAR offers significantly increased sensitivity over
existing methods to detect or isolate rare cells. The detection
components of eDAR can detect a single photon emanated within the
entire aliquot and thus no bioparticle exhibiting detectable
characteristic is missed. The rate of false-negative is extremely
low because aliquot ranking is configured to eliminate only
aliquots that are ranked to be completely devoid of desired
bioparticles.
[0040] Embodiments in accordance with the present invention may be
used in a wide variety of applications in biology and diagnosis of
disease, including capturing cancer cells or cancer stem cells from
body fluids for cancer prognosis; parasites such as giardia or
cryptosporidium for water quality monitoring; malaria-infected
erythrocytes for malaria diagnosis; lymphocytes and leucocytes for
HIV monitoring; fetal cells in maternal blood for disease
screening; stem cells for therapy; prion-infected cells for
prion-related (e.g. mad cow) disease screening.
[0041] In addition to malaria, the present subject matter can be
used for monitoring of CD4+T-lymphocytes (CD4+T-cells) in Human
Immunodeficiency Virus (HIV) diagnostic and monitoring. The
absolute CD4+T-lymphocyte count can serve as a criterion to
initiate antiretroviral therapy and opportunistic infection
prophylaxis in HIV-infected patients. The reduction of
CD4+T-lymphocytes, which is a subpopulation of leucocytes (white
blood cells), strongly correlates to the decline of the
immunological defense. Monitoring of CD4+T-lymphocytes
(CD4+T-cells) level every 3-6 months in all HIV-infected persons
has been recommended by the CDC Public Health Service as a way to
initiate appropriate treatment strategies and to evaluate treatment
efficacy.
[0042] In some laboratories, the absolute CD4+T-cell number is
established using the product of three laboratory techniques: the
total white blood cell count, the percentage of white blood cells
that are lymphocytes, and the percentage of lymphocytes that are
CD4+T-cells. Single platform flow cytometers such as FACSCount (BD
Biosciences) are commercially unavailable in developing countries
or as a portable device. Embodiments according to the present
subject matter can be used to rapidly distinguish CD4+T-lymphocytes
from other leucocytes and RBCs.
[0043] Additional possible applications for separation,
concentration, and/or isolation addressed by embodiments in
accordance with the present invention, include fetal cell
monitoring in maternal blood for prenatal diagnostic of genetic
disorders and prion detection. A prion includes a small infectious
proteinaceous particle which resists inactivation by procedures
that modify nucleic acids. In addition, embodiments according to
the present subject matter can be used with fetal cells or other
micro-biological particulates or nano-biological particulates.
[0044] II. Definitions
[0045] As used herein, a "fluid sample" refers to any liquid that
may or may not contain a rare particle of interest. In certain
embodiments, the fluid sample may be a biological fluid sample, for
example a blood sample, plasma sample, saliva sample, urine sample,
lymph sample, spinal fluid sample, and the like. In other
embodiments, the sample may be an environmental fluid sample, for
example from a lake, river, ocean, pond, stream, spring, marsh,
reservoir, or the like. In yet other embodiments, the sample may be
a water sample, for example from a desalinization plant, water
treatment plant, reservoir, spring, stream, glacial water flow,
water tower, or other water source that may be contemplated as a
source of potable water.
[0046] As used herein, a "rare particle" refers to a cell or
macromolecule present in a fluid sample at a low level. In certain
embodiments, a rare particle may be a cell, protein, protein
complex, nucleic acid, nucleoprotein complex, carbohydrate,
metabolite, catabolite, and the like. In certain embodiments, a
particle may be considered rare if it is present in a fluid sample
at a concentration of less than about 10% of the total particle
population in the fluid, or at less than about 9%, 8%, 7%, 6%, 5%,
4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less of the total
particle population in the fluid. In yet other embodiments, the
rare particle may be present in a fluid sample at less than about 1
part per 10.sup.3 of the total particle population in the fluid, or
at less than about 1 part per 10.sup.4, 10.sup.5, 10.sup.6,
10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, or
less of the total particle population in the fluid.
[0047] In a particular embodiment, the rare particle is a rare
cell. Rare cells may be nucleated or non-nucleated. Rare cells
include, but are not limited to, cells expressing a malignant
phenotype; fetal cells, such as fetal cells in maternal peripheral
blood, tumor cells, such as tumor cells which have been shed from
tumor into blood or other bodily fluids; cells infected with a
virus, such as cells infected by HIV, cells transfected with a gene
of interest; and aberrant subtypes of T-cells or B-cells present in
the peripheral blood of subjects afflicted with autoreactive
disorders.
[0048] As used herein, an "ensemble-decision" refers to a decision
made based on the detection of the presence or absence of a
characteristic in an ensemble, or a group, of particles. In certain
embodiments, an ensemble-decision will be made based on the
presence or absence of a single distinct particle in an aliquot of
a fluid sample containing a plurality of particles. Importantly,
ensemble-decisions made based on the presence or absence of a
single particle will be applied to the entire aliquot (i.e., to all
of the particles present in the aliquot).
[0049] As used herein, an "aliquot" refers to a portion of the
total volume of a fluid sample to be analyzed. An aliquot occupies
a three-dimensional space and the particles within distribute
randomly without organization. An aliquot has a finite depth, and
particles may distribute along the depth with no discernible
layers. In the context of the present invention, an aliquot is
analyzed in its entirety without sub-division. Sheet, ribbon, plane
or similar terms suggesting two-dimensional spaces and used to
describe current cell sorting methods (e.g., flow cytometry, FACS,
etc.) that typically employ hydrodynamic focusing are not
considered an aliquot.
[0050] In certain embodiments, an aliquot may consist of a fraction
of a larger fluid sample, for example, about 1/2 of a fluid sample,
or about 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10, or less of a
fluid sample. In certain embodiments, an aliquot may consist of,
for example, about 10% of a fluid sample, or about 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.001%, or less of a
fluid sample. As such, a fluid that is to be examined or processed
by an eDAR methodology provided herein may be divided, for example,
into at least about 2 aliquots, or at least about 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,
50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200, 225,
250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100,
1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000,
2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000,
9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000,
80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000,
600,000, 700,000, 800,000, 900,000, 1 million, 2 million, 3
million, 4 million, 5 million, 6 million, 7 million, 8 million, 9
million, 10 million, or more aliquots. One of skill in the art
would understand that the number of aliquots into which a fluid
sample would be partitioned into will depend upon the number of
rare particles expected in the fluid and the total volume of the
fluid sample.
[0051] In certain embodiments, an aliquot may have a volume, for
example, of between about 0.1 nL and about 10 mL, or between about
1 nL and about 1 mL, or between about 1 nL and about 100 .mu.L, or
between about 1 nL and about 10 .mu.L, or between about 1 nL and
about 1 .mu.L, or between about 1 nL and about 100 nL.
[0052] As used herein, the term "ranking" refers to assessing a
quantitative property, qualitative property, or importance of an
aliquot by categorization. In one embodiment, an aliquot may be
ranked as either null (for example, when a rare particle is not
detected in the aliquot) or nonzero (for example, when at least one
rare particle is detected in an aliquot). In one embodiment, the
ranking may be binary. In other embodiments, an aliquot may be
ranked according to additional categories, for example, which
correlate with the concentration of the rare particle in the
aliquot, the identity of the rare particle in the aliquot, the
identities of a plurality of different rare particles in the
aliquot, and the like. In this fashion, any number of categories
may be assigned based on ranges of concentration, for example,
between about 1 and 10, between about 11 and 20, between about 1
and 50, between about 51 and 100, between about 1 and 100, between
about 101 and 201, etc. These rankings may be assigned an arbitrary
number corresponding to one of a number of predetermined
quantitative or qualitative categories (e.g., 0, 1, 2, 3, 4, 5,
etc.), or a number corresponding to an actual value for the number
or approximate number or rare particles in the aliquot.
[0053] As used herein, a "detectable characteristic" refers to a
property associated with a rare particle, for example, a
photoactive, electroactive, bioactive, or magnetic property that is
intrinsic to the rare particle or which is associated with a
detectable moiety bound to or conjugated to the rare particle.
[0054] Examples of photoactive properties include, for example,
alterations in optical intensity (optical reflection, scattering,
deflection, transmission, or absorbance) commonly induced by
bioparticle morphology (particle size, granularity, internal
subcellular structures), fluorescence, immunofluorescence, and the
like.
[0055] Examples of electroactive properties include, for example,
changes in the electrical charge, oxidation state, spin state,
capacitance, conductance, dielectric properties, electrophoretic
mobility, or polarizability.
[0056] Examples of bioactive properties include, for example,
detectable interactions with enzymes such as alkaline phosphatase
(AP), horseradish peroxidase (HRP), 62 -Galactosidase and their
chemiluminescent, colometric, or chemifluorescent substrates, which
include but are not limited to TMB
(3,3',5,5'-Tetramethylbenzidine), OPD (o-phenylene Diamine, ABTS
(2,2'-azinodiethylbenzthiazoline sulfonate), chlornaphthol, AEC
(3-amino-9-ethylcarbazole), DAB (Diaminobenzidine), pNPP
(p-Nitrophenyl Phosphate), BCIP/NBT (Bromochloroindolyl
Phosphate-Nitro blue Tetrazolium, and the like.
[0057] In certain embodiments, moieties that can be used to detect
a rare particle include, without limitation, nanoparticles,
microbeads, antibodies and fragments thereof, fluorescent
antibodies, magnetic nanoparticles, polymer molecules, dye
molecules, DNA or RNA molecules (e.g. aptamers), lipid molecules,
protein molecules, and the like.
[0058] As used herein an "antibody" refers to a polypeptide
comprising a framework region from an immunoglobulin gene or
fragments thereof that specifically binds and recognizes an
antigen. The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon, and mu constant region genes,
as well as the myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively. Typically, the antigen-binding region of an antibody
will be most critical in specificity and affinity of binding.
Antibodies can be polyclonal or monoclonal, derived from serum, a
hybridoma or recombinantly cloned, and can also be chimeric,
primatized, or humanized.
[0059] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (VL) and variable heavy chain (VH) refer to
these light and heavy chains respectively.
[0060] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2 a dimer of Fab which itself is a light chain joined to
V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially Fab with part of the
hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).
While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by
using recombinant DNA methodology. Thus, the term antibody, as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., Nature, 348:552-554 (1990)).
[0061] In one embodiment, the antibody is conjugated to a label or
detectable moiety.
[0062] As used herein, a "label" or a "detectable moiety" refers to
a composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, chemical, or other physical means. For
example, useful labels include, without limitation radionuclides,
fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate
(FITC), Oregon Green.TM., rhodamine, Texas red, tetrarhodimine
isothiocynate (TRITC), Cy3, Cy5, etc.), fluorescent markers (e.g.,
green fluorescent protein (GFP), phycoerythrin, etc.), autoquenched
fluorescent compounds that are activated by tumor-associated
proteases, enzymes (e.g., luciferase, horseradish peroxidase,
alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin,
and the like.
[0063] In certain embodiments, detection reagents may be perfused
to selectively label or accentuate the isolated cells. Examples of
such reagents include, without limitation, fluorescent,
immunofluorescent, dye-conjugated molecules (such as antibodies,
fab fragments, aptamers, polymers, ligands, agonists, antagonists,
or combinations thereof) magnetic, electroactive, bioactive, or
photoactive compounds. An example is to use a stain that reacts
with cytokeratins, which are integral components of the
cytoskeleton in epithelial cancerous cells. Other dye examples
include fluorescein isothiocyanate (FITC)-conjugated mouse
anti-human epithelial antibody (HEA) and phycoerythrin
(PE)-conjugated anti-CD45. Other examples of dye-conjugated
antibodies include but are not limited to the pan-cytokeratin
antibody A45B/B3, AE1/AE3, or CAM5.2 (pan-cytokeratin antibodies
that recognize Cytokeratin 8 (CK8), Cytokeratin 18 (CK18), or
Cytokeratin 19 (CK19) and ones against: breast cancer antigen
NY-BR-1 (also known as B726P, ANKRD30A, Ankyrin repeat domain 30A);
B305D isoform A or C (B305D-A ro B305D-C; also known as antigen
B305D); Hermes antigen (also known as Antigen CD44, PGP1);
E-cadherin (also known as Uvomorulin, Cadherin-1, CDH1);
Carcino-embryonic antigen (CEA; also known as CEACAM5 or
Carcino-embryonic antigen-related cell adhesion molecule 5);
.beta.-Human chorionic gonadotophin (.beta.-HCG; also known as CGB,
Chronic gonadotrophin, .beta.polypeptide); Cathepsin-D (also known
as CTSD); Neuropeptide Y receptor Y3 (also known as NPY3R;
Lipopolysaccharide-associated protein3, LAP3, Fusion; Chemokine
(CXC motif, receptor 4); CXCR4); Oncogene ERBB1 (also known as
c-erbB-1, Epidermal growth factor receptor, EGFR); Her-2 Neu (also
known as c-erbB-2 or ERBB2); GABA receptor A, pi (.pi.) polypeptide
(also known as GABARAP, GABA-A receptor, pi (.pi.) polypeptide
(GABA A(.pi.), .gamma.-Aminobutyric acid type A receptor pi (.pi.)
subunit), or GABRP); ppGalNac-T(6) (also known as
.beta.-1-4-N-acetyl-galactosaminyl-transferase 6, GalNActransferase
6, Ga1NAcT6, UDP-N-acetyl-d-galactosamine:polypeptide
N-acetylgalactosaminyltransferase 6, or GALNT6); CK7 (also known as
Cytokeratin 7, Sarcolectin, SCL, Keratin 7, or KRT7); CK8 (also
known as Cytokeratin 8, Keratin 8, or KRT8); CK18 (also known as
Cytokeratin 18, Keratin 18, or KRT18); CK19 (also known as
Cytokeratin 19, Keratin 19, or KRT19); CK20 (also known as
Cytokeratin 20, Keratin 20, or KRT20); Mage (also known as Melanoma
antigen family A subtytpes or MAGE-A subtypes); Mage3 (also known
as Melanoma antigen family A 3, or MAGA3); Hepatocyte growth factor
receptor (also known as HGFR, Renal cell carninoma papillary 2,
RCCP2, Protooncogene met, or MET); Mucin-1 (also known as MUC1,
Carcinoma Antigen 15.3, (CA15.3), Carcinoma Antigen 27.29 (CA
27.29); CD227 antigen, Episialin, Epithelial Membrane Antigen
(EMA), Polymorphic Epithelial Mucin (PEM), Peanut-reactive urinary
mucin (PUM), Tumor-associated glycoprotein 12 (TAG12)); Gross
Cystic Disease Fluid Protein (also known as GCDFP-15,
Prolactin-induced protein, PIP); Urokinase receptor (also known as
uPR, CD87 antigen, Plasminogen activator receptor urokinase-type,
PLAUR); PTHrP (parathyroid hormone-related proteins; also known as
PTHLH); BS106 (also known as B511S, small breast epithelial mucin,
or SBEM); Prostatein-like Lipophilin B (LPB, LPHB; also known as
Antigen BU101, Secretoglobin family 1-D member 2, SCGB1-D2);
Mammaglobin 2 (MGB2; also known as Mammaglobin B, MGBB, Lacryglobin
(LGB) Lipophilin C (LPC, LPHC), Secretoglobin family 2A member 1,
or SCGB2A1); Mammaglobin (MGB; also known as Mammaglobin 1, MGB1,
Mammaglobin A, MGBA, Secretoglobin family 2A member 2, or SCGB2A2);
Mammary serine protease inhibitor (Maspin, also known as Serine (or
cystein) proteinase inhibitor Glade B (ovalbumin) member 5, or
SERPINB5); Prostate epithelium-specific Ets transcription factor
(PDEF; also known as Sterile alpha motif pointed domain-containing
ets transcription factor, or SPDEF); Tumor-associated calcium
signal transducer 1 (also known as Colorectal carcinoma antigen
CO17-1A, Epithelial Glycoprotein 2 (EGP2), Epithelial glycoprotein
40 kDa (EGP40), Epithelial Cell Adhesion Molecule (EpCAM),
Epithelial-specific antigen (ESA), Gastrointestinal
tumor-associated antigen 733-2 (GA733-2), KS1/4 antigen, Membrane
component of chromosome 4 surface marker 1 (M4S1), MK-1 antigen,
MIC18 antigen, TROP-1 antigen, or TACSTD1); Telomerase reverse
transcriptase (also known as Telomerase catalytic subunit, or
TERT); Trefoil Factor 1 (also known as Breast Cancer
Estrogen-Inducible Sequence, BCEI, Gastrointestinal Trefoil
Protein, GTF, pS2 protein, or TFF1); folate; or Trefoil Factor 3
(also known as Intestinal Trefoil Factor, ITF, pl.B; or TFF3).
[0064] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a rare particle, for example a protein, nucleic
acid, or cell, refers to a binding reaction that is determinative
of the presence of the rare particle, often in a heterogeneous
population of particles and other biologics. Thus, under designated
immunoassay conditions, the specified antibodies bind to a
particular rare particle at least two times the background and more
typically more than 10 to 100 times background. Specific binding to
an antibody under such conditions requires an antibody that is
selected for its specificity for a particular particle. For
example, polyclonal antibodies can be selected to obtain only those
polyclonal antibodies that are specifically immunoreactive with the
selected antigen and not with other proteins. This selection may be
achieved by subtracting out antibodies that cross-react with other
molecules.
[0065] In one aspect, the present invention provides a method for
detecting one or more rare bioparticles in a sample fluid; said
method comprising: a) interrogating one or more aliquots of said
sample fluid; b) in a single measurement detecting presence or
absence of said one or more rare bioparticles in each of said one
or more aliquots wherein at least one of said one or more aliquots
comprises multiple bioparticles; and c) ranking said aliquots based
on the presence or absence of said one or more rare bioparticles.
In certain embodiments, the rare bioparticles are cells. In a
certain embodiment, the rare bioparticles are fluorescent labeled
cells.
[0066] In some embodiments of the methods provided herein, multiple
parameters are detected in a single measurement. In a particular
embodiment, the multiple parameters are different fluorescent
colors.
[0067] In certain embodiments of the methods provided herein, the
sample fluid is stabilized by addition of anticoagulants, compounds
that prevent agglomeration of cells in the sample including said
bioparticles or their combinations.
[0068] In some embodiments, the aliquot ranking is binary, for
example an aliquot is assigned a value of "0" if the aliquot does
not contain a rare article and a value of "1" if it does. In other
embodiments, the ranking is non-binary, for example, the value is
assigned based on the number or rare particles present in the
aliquot or the identity of the rare particles in the sample. In
certain embodiments, the ranking is performed by a computer and a
software representing a ranking algorithm.
[0069] In some embodiments, the methods provided herein further
comprise a step of channeling the aliquots based on their ranking
For example, the flow or collection of the aliquots is directed
based on the value assigned to the aliquot. In certain embodiments,
this is achieved by the use of external fields or by creating flow
disturbances.
[0070] In certain embodiments, the method may comprise
concentrating the rare bioparticles by collecting and/or pooling
aliquots with similar said ranking
[0071] In some embodiments, of the methods provided herein, the
rare bioparticles are selected from the group consisting of cancer
cells, cancer stem cells, giardia, cryptosporium, malaria infected
erythrocytes, lymphocytes, leucocytes, fetal cells, stem cells and
prion-infected cells.
[0072] In another aspect, the present invention provides a device
for detecting one or more rare bioparticles in a sample fluid; said
device comprising: a) one or more detectors for detecting presence
or absence of one or more rare bioparticles in each of the one or
more aliquots wherein at least one of said one or more aliquots
comprises multiple bioparticles; and b) a computer with software
for ranking said aliquots based on presence or absence of said one
or more rare bioparticles. In one embodiment, the ranking is
binary. In other embodiments, wherein the device is used to detect
multiple types of bioparticles, the ranking is non-binary.
[0073] In certain embodiments, the device may further comprise
channels for channeling said aliquots based on said ranking In
particular embodiments, the channels are treated with anticoagulant
compounds, compounds that preferentially bind to the rare
bioparticles, compounds that prevent bioparticles agglomeration or
their combinations.
[0074] In certain embodiments, the device may further comprise
electrodes for tracking and manipulating the trajectory of said
bioparticles. In other embodiments, the device may further comprise
magnetic elements for the separation of bioparticles with attached
magnetic particles. In et other embodiments, the device may further
comprise acoustical elements for tracking and manipulating the
trajectory of said bioparticles.
[0075] In certain embodiments of the devices and apparatuses
provided herein, the device comprises one or more detectors are
selected from a camera, an electron multiplier, a charge-coupled
device (CCD) image sensor, a photomultiplier tube (PMT), an
avalanche photodiode (APD), a single-photon avalanche diode (SPAD),
or a complementary metal oxide semiconductor (CMOS) image
sensor.
[0076] In certain embodiments of the devices and apparatuses
provided herein, the device may further comprise one or more
sources for interrogating one or more aliquots of said sample
fluid. For example, a source of electromagnetic radiation. In
particular embodiments, the one or more sources for interrogating
are selected from, a laser (solid state, diode-pumped, ion, or
dye), a light-emitting diode (LED), a lamp, an arc discharge, a
magnetic pulse, or a natural light. In yet other embodiments, a
source for interrogation of the aliquot is not required when the
bioparticle exhibits light emission such as chemiluminescence or
bioluminescence.
[0077] III. Embodiments
[0078] A. Detection Methods
[0079] In one aspect, the present invention provides a method for
detecting a rare particle in a fluid sample, the method comprising
the steps of: (a) detecting the presence or absence of the rare
particle in an aliquot of the fluid sample; (b) assigning a value
to the aliquot based on the presence or absence of the rare
particle; and (c) directing the flow or collection of the aliquot
based on the assigned value.
[0080] In one embodiment, the step of detecting the presence of the
rare particle comprises the sub-steps of: (i) contacting the fluid
sample with a detection reagent under conditions suitable to
transform the detection reagent into a complex comprising said
detection reagent and a rare particle; and (ii) detecting the
presence or absence of a complex formed in step (i) in an aliquot
of the fluid sample.
[0081] In certain embodiments, the detection reagent may comprise a
labeled or unlabeled antibody, fab fragment, aptamer, polymer,
nanoparticle, microbead, fluorescent antibody, magnetic
nanoparticle, polymer molecule, dye molecule, aptamer, lipid
molecule, protein molecule, and the like.
[0082] In another embodiment, the step of detecting the presence of
the rare particle comprises the sub-steps of: (i) interrogating the
aliquot with an external source of electromagnetic radiation; and
(ii) detecting fluorescence of the rare particle.
[0083] In one embodiment, the rare particle may comprise a
fluorescently labeled cell. In a certain embodiment, the
fluorescently labeled cell may comprise a cell that expresses a
fluorescent protein, or a cell that has been labeled with a
fluorescent detection reagent. For example, a cell that transiently
or stably expresses a red or green fluorescent protein.
[0084] In yet another embodiment, wherein the rare particle
exhibits intrinsic chemiluminescence or bioluminescence, the step
of detecting the presence of a rare particle comprises detecting
bioluminescence or chemiluminescence of the rare particle.
[0085] In certain embodiments, the rare particle may be a cell,
protein, protein complex, nucleic acid, nucleoprotein complex,
carbohydrate, metabolite, catabolite, and the like. In one
embodiment, the rare particle is a cell. In particular embodiments,
the cell may be a cancer cell, a circulating tumor cell (CTC), a
cancer stem cell, a cancer cell displaying a cancer surface
antigen, for example, one selected from the groups consisting of
CD44, CD2, CD3, CD10, CD14, CD16, CD24. CD31, CD45, CD64, CD140b,
or a combination thereof.
[0086] Cancer stem cells may be distinguished from ordinary cancer
cells by perfusing other reagents that selectively bind to
biomarkers, which may include but are not limited to CD44, CD2,
CD3, CD10, CD14, CD16, CD24. CD31, CD45, CD64 or CD140b.
[0087] In certain embodiments, wherein the rare particle is a
cancer cell, cells contained within the aliquots identified as
having a rare cell may be further individually dissected. For
example, these cells may be further partitioned or sorted via
traditional flow cytometry or eDAR and desired cells may be
dissected to understand the origin of malfunctioning cellular
machinery. The contents within each cell may be individually
analyzed for DNA, RNA, DNA sequence, metabolite, lipid,
carbohydrate, protein content, or the like.
[0088] In other embodiments, the rare cell may be a parasitic cell
or organism, for example, a species of Giardia or Cryptosporidium,
a erythrocyte infected with a species of Plasmodium, a lymphocyte
or leucocyte infected with HIV, a fetal cell in maternal blood, a
stem cell, a prion-infected cell, a CD4+T-cell, and the like.
[0089] In one embodiments, the fluid sample may comprise more than
one type of rare particle, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more types of rare particles. Accordingly, in certain
embodiments, the fluid sample is simultaneously contacted with a
plurality of differentiable detection reagents each having a
different specificity under conditions sufficient to transform the
plurality of detection reagents into a plurality of complexes
comprising the detection reagents and a plurality of rare
particles. In some embodiments, the plurality of complexes are
detected simultaneously, for example, by using an eDAR apparatus
that comprises more than one interrogation devices and/or more than
one detection devices.
[0090] For example, in the case that two rare particles are to be
detected simultaneously, each rare particle may be contacted with a
differentiable detection reagent, each of which may be detected by
one of two detection devices. Furthermore, wherein the detection
reagents comprise fluorescent moieties, two interrogation devices
(e.g., two lasers producing radiation at different wavelengths
corresponding to excitation wavelengths of the different
fluorescent moieties) may be used and the respective fluorescent
radiation may be detected by two different detection devices.
Accordingly, in one embodiment, the detection reagents are
differentiable by fluorescence at different wavelengths.
[0091] In yet another embodiment, the two or more rare particles
may be detected in series. For example, in one embodiment, the
method may comprise detecting a first rare particle at a first
location of an eDAR apparatus and detecting a second rare cell at a
second location of an eDAR apparatus. In this fashion, the aliquot
in which the first and second particle reside may be channeled
after the first detection step, after the second detection step, or
after both detection steps.
[0092] In certain embodiments of the invention, detection of a
characteristic from an ensemble of cells can be simultaneous or
cumulative over time. For example, detection of a characteristic
can emanate at once ("simultaneous") from a large aliquot
containing an ensemble of bioparticles.
[0093] In certain embodiments, in which the method is performed in
a simultaneous mode, the bioparticles may be carried by a flow of
variable velocity. As an example, bioparticles may be carried by a
steady flow as they traverse through the detection volume.
Alternatively, the flow may be stopped, decelerated, or accelerated
as the cells traverse through the detection volume. Flow may be
regulated with one of the following either upstream or downstream
of the detection volume: a valve, a bubble, an electric field, a
magnetic field, an optical field, a pneumatic pressure source, a
solid particle, a membrane, an immiscible droplet, a gravitational
differential, or a coating to alter surface tension of the
channel.
[0094] In some embodiments of the methods provided herein, the
detection step is performed during continuous flow of the fluid
sample through a flow channel. In certain embodiments, the
individual aliquots are not physically separated, but rather are
defined by the optical detection step, i.e., an aliquot may be
defined as the ensemble of particles present in the detection
volume at the instant the detection occurs.
[0095] In certain embodiments, the detection event will occur with
a regular frequency, which is dependent upon both the size of the
detection volume and the flow rate of the fluid sample. For
example, if the detection volume of a particular apparatus is 10
.mu.L, and the fluid sample is flowed through the apparatus at a
rate of 100 .mu.L/second, a different aliquot will be detected
every 0.1 seconds, or at a rate of 10 Hz.
[0096] In certain embodiments, dependent upon the geometry of the
apparatus and the volume of the fluid to be processed, discrete
aliquots traverse through the detection volume at a rate between
0.1 kHz and 100 MHz. In another embodiment, the discrete aliquots
traverse through the detection volume at a rate between about 10 Hz
and about 10 MHz. In other embodiments, the discrete aliquots may
traverse through the detection volume at a frequency of between
about 0.1 kHz and about 100 MHZ, or between about 1 kHz and about
10 MHz, or between about 1 kHz and about 5MHz, or between about 1
kHz and about 1 MHz. In certain embodiments, the frequency by which
the aliquots traverse through the detection volume may be at least
about 0.1 kHz, or at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,
60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 400, 500, 600, 700,
800, or 900 kHz, or at least about 1 MHz, or at least about 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,
90, or 100 MHz.
[0097] In other embodiments of the methods provided herein,
detection of a characteristic from an ensemble of cells can emanate
over time ("cumulative") from a small detection volume which is on
the order of a single cell, but with multiple cells traversing
through the detection volume with the aid of flow. Cumulative mode
of eDAR is distinct from time-lapse overlay of consecutive signals
or frames emanating from a single bioparticle; timelapse overlay of
a single bioparticle does not constitute an ensemble of
bioparticles. In both simultaneous and cumulative, a decision is
rendered only after a characteristic from an ensemble of cells has
been detected.
[0098] In yet other embodiments of the methods provided herein, the
aliquots may be physically separated prior to detection. This may
be accomplished, for example, by partitioning the sample fluid into
discrete aqueous aliquots separated by air or a continuous
oil-immiscible fluid phase, for example, into a droplet.
[0099] In one embodiment, the immiscible phase used to separate
aqueous aliquots may include an organic phase, an oil, natural oils
such as mineral oil and soybean oil, silicone oils such as AR-20,
AS-4, PDMS oil, fluorinated oils such as Fluorinert and
perfluorordecalin, organic solvents such as hexadecane and
acetophenone, a wax, air, or gas.
[0100] In certain embodiments, an immiscible phase may be
continuous (i.e. surrounds the discrete aliquots entirely) or
segmented (i.e. occupies only the spacing between discrete aliquots
but does not completely surround the aliquots).
[0101] For example, FIG. 16 illustrates a an immiscible phase
(1642) that surrounds the discrete aliquots entirely.
[0102] In one embodiment, the discrete aliquots are droplets. In
another embodiment, the discrete aliquots are plugs.
[0103] In one embodiment, the discrete aliquots may be formed
sequentially on an eDAR apparatus in a flow channel in fluidic
communication with the flow channel.
[0104] In certain embodiments, the discrete aliquots may be formed
externally of the eDAR apparatus but flowed into a flow channel of
the apparatus via a tubing, a port, or an interconnect in fluidic
communication with the flow channel.
[0105] In one embodiment, surfactants may be added to the cell
suspension or the immiscible phase to stabilize the discrete
aliquots. Surfactants may include albumin (bovine or human serum
albumin), Span 80, Pluronic, octaethylene glycol monodecyl ether,
tetraethylene glycol monodecyl ether, zwitterionic surfactants such
as N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (DDAPS),
anionic surfactants such as sodium dioctyl sulfosuccinate (AOT),
cationic surfactants such as cetyl trimethyl ammonium bromide
(CTAB), and silicone-based, PEGylated and fluorinated
surfactants.
[0106] In yet another embodiment, the fluid sample may be
partitioned into aliquots and physically separated into separate
flow channels or chambers of an eDAR apparatus prior to the
detection step. The subsequent detection step may then be performed
either in parallel (i.e., at the same time using multiple detection
devices or a single detection device), or sequentially, for example
by directing the individual aliquots sequentially through one or
more detection volumes.
[0107] In some embodiments of the invention, the fluid sample, for
example a biological fluid sample, may be stabilized prior to
detection of a rare particle. In certain embodiments, the fluids
may be stabilized with a reagents, including but not limited to, an
anticoagulant such as citrate, heparin, ethylenediamine tetraacetic
acid (EDTA), diethylenetriamine pentaacetic acid (DTPA),
1,2-diaminocyclohexane tetraacetic acid (DCTA), or ethylene
bis(oxyethylenenitrilo) tetraacetic acid (EGTA); an aldehyde such
as methylol, hydroxymethyl derivatives of amines or amides of
formaldehyde, diazolinidinyl urea, imidazolidinyl urea,
methenamine, paraformaldehyde, glutaraldehyde, or glyoxal, and the
like.
[0108] In yet other embodiments of the invention, the methods
provided herein may be further coupled to a secondary process
occurring after channeling of the desired aliquots. Example of
processes and/or functions that may be coupled to a method provided
herein include, for example, selective reactions to identify
cellular contents (e.g. DNA, RNA, microRNA, lipids, metabolites,
carbohydrates, or proteins encapsulated within cells). These
reactions include Polymerase Chain Reaction (PCR), Real-Time
Polymerase Chain Reaction (RT-PCR), isothermal PCR, reactions to
determine the epigenetic states of DNA, single-molecule
hybridization reactions to determine the microRNA and siRNA
contents, or aptamer (short strands of DNA)-selective
reactions.
[0109] In certain embodiments, the methods provided herein may
further be coupled to an assay protocol following aliquot or cell
isolation. Non-limiting examples of assays that may be coupled to
the methods provided herein include nucleic-acid based methods such
as RNA extraction (with or without amplification), cDNA synthesis
(reverse transcription), gene microarrays, DNA extraction,
Polymerase Chain Reactions (PCR) (single, nested, quantitative
real-time, or linker-adapter), or DNA-methylation analysis;
cytometric methods such as fluorescence in situ hybridization
(FISH), laser capture microdissection, flow cytometry, fluorescence
activated cell sorting (FACS), cell culturing, or comparative
genomic hybridization (CGH) studies; chemical assay methods such as
electrophoresis, Southern blot analysis or enzyme-linked
immunosorbent assay (ELISA); assays to determine the microRNA and
siRNA contents; assays to determine the DNA/RNA content; assays to
determine lipid contents; assays to determine carbohydrate
contents; assays to determine metabolite contents; assays to
determine protein contents; and functional cell assays (e.g.
apoptotic assays, cell migration assays, cell proliferation assays,
cell differentiation assays, etc.), and the like.
[0110] In yet another embodiment, the methods provided herein may
further be coupled to flow cytometry, for example, to further
partition or isolate rare particles present in a selected aliquot.
In one embodiment, a channel of the eDAR device used for the
methods provided herein may be in fluidic communication with a flow
cytometer. In certain embodiments, the coupling of eDAR and flow
cytometry allows for selected aliquots to be further examined or
serially sorted to further enrich a population of rare particles or
cells. In certain embodiments of the methods provided herein, this
configuration allows for upstream gross-sorting of rare particles
or cells and only directs aliquots containing rare particles or
cells into downstream processes, such as flow cytometry, that are
time, cost, and/or labor intense.
[0111] In certain embodiments, the methods provided herein may be
performed using an eDAR apparatus provided herein.
[0112] 1. Advantageous Features
[0113] As noted above, due in part to the ensemble detection and
ranking of whole aliquots, rather than individual cells or
particles, eDAR technologies are much faster and less expensive
than traditional flow cytometry methods currently employed. Several
features contribute to the improved eDAR methodologies.
[0114] For example, in one embodiment of the methods provided
herein, an aliquot comprises more than a single particle, cell, or
fluorescent entity. In this regard, discreet volumes containing a
plurality of cells or particles, rather than single cells or
particles, or 1 dimensional sheets of cells or particles, can be
interrogated simultaneously.
[0115] In a related embodiment of the methods provided herein, the
particles or cells of a fluid do not need to enter the detection
spot or volume serially (i.e., one after another without
overlapping presence). In a related embodiment, the particles do
not need to enter the detection spot or volume in a single row or
sheet. Accordingly, in one embodiment of the methods provided
herein, multiple bioparticles, within a single aliquot, may pass
through a cross-section or cross-sectional volume of a flow
channel, for example an interrogation and/or detection volume, at a
time.
[0116] In one embodiment of the methods provided herein, a sheath
flow, guiding buffer, or other hydrodynamic or geometric focusing
mechanisms is not needed to focus the bioparticles into a single
row for interrogation and/or detection.
[0117] In one embodiment of the methods provided herein, a ranking
scheme or value assignment scheme of complete aliquots is employed,
instead of sorting individual bioparticles.
[0118] In one embodiment of the methods provided herein, an
ensemble of particles or cells is detected simultaneously, for
example as a single aliquot of a larger fluid sample. In a related
embodiment, a decision made for an aliquot affects the entire
ensemble of particles or cells contained within the aliquot. In yet
another related embodiment, an ensemble of particles or cells
detected simultaneously within an aliquot will remain as an
ensemble of particles or cells.
[0119] 2. Aliquot Ranking
[0120] In one embodiment of the methods provided herein, the
aliquot is assigned either a first value is the aliquot contains a
rare particle or a second value if the aliquot does not contain a
rare particle. In a particular embodiment, the ranking (i.e.,
assignment of a value) is binary. For example, each aliquot
containing at least one rare particle is assigned a value of 1,
while each aliquot not containing a rare particle is assigned a
value of 0.
[0121] In another embodiment of the methods provided herein, the
aliquot is assigned a value according to the quantity of rare
particles present in the aliquot. For example, an aliquot
containing 4 rare particles may be assigned a value of 4.
Alternatively, an aliquot containing 4 particles may be assigned a
value that corresponds to a particular range of rare particle
quantities, for example 0 to 5 particles, 1 to 10 particles 4 to 6
particles, etc.
[0122] In yet another embodiment of the methods provided herein,
wherein more than one type of rare particles are present in a
single fluid sample, an aliquot is assigned a value according to
the identities of any rare particles in the aliquot. For example,
wherein a fluid sample contains two rare particles, A and B, an
aliquot containing neither A nor B may be assigned a value of 0, an
aliquot containing only A may be assigned a value of 1, an aliquot
containing only B may be assigned a value of 2, and an aliquot
containing both A and B may be assigned a value of 3. Accordingly,
in one embodiment of the methods provided herein, wherein more than
one type of rare particles are present in a single fluid sample,
the ranking (i.e., assignment of a value) is not binary.
[0123] In certain embodiments, a non-null assigned value may
depends on either the identity of the rare particle or the
concentration of the rare particle.
[0124] In certain embodiments, multiple aliquots having the same
assigned value are pooled or channeled together.
[0125] In one embodiment of the methods of the present invention,
an active decision is required to rank or assign a value to an
aliquot. In certain embodiments, a computer, controller, chip with
integrated circuits, circuit board, electronic element, software,
and/or algorithm is used to rank or assign a value to an
aliquot.
[0126] 3. Aliquot Channeling and Fluid Flow
[0127] In certain embodiments of the present invention, directing
the flow or collection of an aliquot is based on the value assigned
to the aliquot. For example, in an embodiment wherein a single type
of rare particle is present in a fluid sample, an aliquot assigned
a null or "0" value may be directed into a first channel
(channeled) or waste outlet and an aliquot assigned a positive or
"1" value may be directed into a second channel or collection
chamber.
[0128] In other embodiments of the present invention, wherein more
than one type of rare particle is present in the fluid sample, an
aliquot may be channeled based on the particular composition of
rare particles present in the aliquot. In one embodiment, an
aliquot containing no rare particles may be directed into a first
channel or waste outlet, an aliquot containing a first type of rare
particle may be directed into a second channel or a first
collection chamber, and an aliquot containing a second type or rare
particle may be directed into a third channel or second collection
chamber.
[0129] In certain embodiments, an aliquot containing more than one
type of rare particle may be directed into a particular flow
channel or collection chamber. Alternatively, the aliquot may be
directed into a mixing or dilution chamber and subsequently the
mixed or diluted aliquot may be further partitioned into
sub-aliquots such that the rare cells are partitioned into
different sub-aliquots. The rare cells in the sub-aliquots may then
be detected again such that the rare cells can be separated from
each other.
[0130] In one embodiment of the methods provided herein, the step
of channeling (i.e., directing the flow or collection of the
aliquots may be performed by the use of external fields or by
creating flow disturbances.
[0131] In one aspect of the invention, once an aliquot is ranked,
external fields may be used to alter the aliquot direction. The
fields may include electric field, magnetic field, electrokinetic,
electrophoretic, dielectrophoretic, hydrodynamic, gravitational,
pneumatic or optical forces. Alternatively external flow
disturbances may be induced with an introduction of materials
immiscible with cell suspension, such as air, immiscible organic
liquid, or microbeads.
[0132] In certain embodiments, the flow can be delivered by, for
example, methods and devices that induce hydrodynamic fluidic
pressure, which includes but is not limited to those that operate
on the basis of mechanical principles (e.g. external syringe pumps,
pneumatic membrane pumps, vibrating membrane pumps, vacuum devices,
centrifugal forces, and capillary action); electrical or magnetic
principles (e.g. electroosmotic flow, electrokinetic pumps
piezoelectric/ultrasonic pumps, ferrofluidic plugs,
electrohydrodynamic pumps, and magnetohydrodynamic pumps);
thermodynamic principles (e.g. gas bubble
generation/phase-change-induced volume expansion); surface-wetting
principles (e.g. electrowetting, chemically, thermally, and
radioactively induced surface-tension gradient); and the like.
[0133] In yet other embodiments, the fluid can be delivered or
channeled by a fluid drive force provided by gravity feed, surface
tension (like capillary action), electrostatic forces
(electrokinetic flow), centrifugal flow (substrate disposed on a
compact disc and rotated), magnetic forces (oscillating ions causes
flow), magnetohydrodynamic forces and a vacuum or pressure
differential.
[0134] In certain embodiments, Fluid flow control devices, such as
those enumerated with regard to methods and devices for inducing
hydrodynamic fluid pressure or fluid drive force, can be coupled to
an input port or an output port of the present subject matter. In
one example, multiple ports are provided at either or both of the
inlet and outlet and one or more ports are coupled to a fluid flow
control device.
[0135] B. Diagnostic and Prognostic Methods
[0136] In one aspect, the present invention provides a method for
providing a subject a diagnosis or prognosis for a condition
associated with the presence of a rare particle in a fluid sample,
for example a biological fluid such as a blood sample.
[0137] In one embodiment the method comprises the steps of: (a)
detecting the presence or absence of the rare particle in an
aliquot of the biological fluid; (b) assigning a value to the
aliquot based on the presence or absence of the rare particle; and
(c) directing the flow or collection of the aliquot based on the
assigned value.
[0138] In another embodiment, the method comprises the steps of:
(a) contacting a biological fluid from the subject with a detection
reagent under conditions suitable to transform the detection
reagent into a complex comprising said detection reagent and a rare
particle; (b) detecting the presence or absence of a complex formed
in step (a) in an aliquot of the biological fluid; (c) assigning a
value to the aliquot based on the presence or absence of a complex
formed in step (a); and (d) providing a diagnosis or prognosis to
the subject based on the assigned value.
[0139] In another embodiment, the step of detecting the presence of
the rare particle comprises the sub-steps of: (i) interrogating the
aliquot with an external source of electromagnetic radiation; and
(ii) detecting fluorescence of the rare particle.
[0140] For more than 100 years, physicians have known that cancers
spread by shedding cells into the blood. As blood carries these
cancer cells from organ to organ, cancer metastasizes. These loose
tumor cells are called Circulating Tumor Cells (CTC). In one
aspect, the present invention offers accurate methods for counting
and isolating these cancer cells from the peripheral blood.
[0141] Accurate detection of CTCs in blood turns out to be
exceedingly difficult because of the astronomical number of red and
white blood cells also present. With as many as 5 billion red blood
cells and 5 million white blood cells co-existing to mask a single
CTC, the problem of detecting CTCs is literally finding a needle in
a haystack.
[0142] By accurately counting the number of cancer cells in blood,
the present invention offers a real-time snapshot of the cancer
spreading process. The most remarkable differentiation of a CTC
blood test from the traditional prognostic tools (e.g., status of
lymph nodes, tumor size, and morphologic features) is that the CTC
blood test can be used to provide early feedback on whether a
cancer treatment is effective. Patients undergoing the 6-month
chemotherapy may have their CTC counts measured every 3-4 weeks; if
the count remains high, the oncologist may deem the current
treatment ineffective and prescribe new drugs. From the patients'
perspective, having a CTC test can (1) provide a substantial
savings by eliminating ineffective chemotherapy, which can cost
between about $3,000-$10,000/month per drug, (2) grant them
precious opportunities to find an effective treatment before it is
too late. These reasons alone are important enough for oncologists
to routinely prescribe expensive radiological imaging scans (e.g.,
CT or MRI). However, in one aspect, the present invention provides
a rapid, inexpensive CTC test that is cheaper, safer, more
reproducible, and provides the same, if not more accurate,
prognostic information six weeks earlier than a radiological
imaging scan. There is currently no biomarker test available that
offers similar advantages.
[0143] Due to a high sensitivity in cancer cells detected, the
methods described herein provide the potential of detecting cancer
cells before their concentration reach the lower detection limit of
competing technologies. This means that the methods provided herein
are able to yield meaningful results earlier than competing
technologies. Consequently, instead of limiting the use of the
present technology to Stage IV metastatic cancer, oncologists may
expand its use toward early diagnostic (i.e., Stage III, Stage II,
Stage I, or metastatic, or pre-cancerous), for example by
periodically prescribing the use of the methods provided herein to
the general public not yet exhibiting symptoms of cancer. Generally
healthy people do not have any CTCs in blood; if any CTC is
detected in the unsuspecting patients using the present invention,
then further tests, (e.g., CT, MRI,) can be prescribed to locate
the tumors and confirm the status.
[0144] In another embodiment, tumor cells isolated using the
present invention may be further subjected to subpopulation
analysis (e.g., according to genotype or phenotype) to develop a
targeted treatment. As an example, the isolated tumor cells can be
incubated with fluorescent antibodies binding to specific drug
targets to determine the presence or degree of expression of a drug
target. Once the expression of the drug target is confirmed, an
oncologist can be assured to choose from drugs specifically
developed to target the expression. In one example, the isolated
tumor cells may be incubated with fluorescent antibodies binding
specifically to Her2 receptor to determine whether the breast tumor
shedding CTCs is Her2-positive. If the isolated tumor cells exhibit
high Her2 expression, oncologist may prescribe Herceptin
(trastuzumab), since this drug is designed to target and block the
function of HER2 protein overexpression. Other known drug targets,
including BCR-ABL or PDGFR (targeted by drug Gleevec), ERBB2
(targeted by Herceptin), EFGR (targeted by Iressa, Tarceva),
RAR-alpha (targeted by ATRA), Oestrogen receptor (targeted by
Tamoxifen), aromatase (targeted by Letrazole), androgen receptor
(targeted by Flutamide, Biclutamide), CD20 (targeted by Rituximab),
VEGF-receptor (targeted by Avastin) can also be similarly screened
from the isolated tumor cells before prescribing the appropriate
chemotherapy regimen.
[0145] In a specific embodiment, the rare particle is a cancer cell
or circulating tumor cell (CTC). In other embodiments, the rare
cell may be a parasitic cell or organism, for example, a species of
Giardia or Cryptosporidium, a erythrocyte infected with a species
of Plasmodium, a lymphocyte or leucocyte infected with HIV, a fetal
cell in maternal blood, a stem cell, a prion-infected cell, a
CD4+T-cell, and the like.
[0146] In one embodiment, a method for diagnosing malaria is
provided, the method comprising detecting an erythrocyte infected
with Plasmodium using an eDAR method and/or apparatus provided
herein.
[0147] In another embodiment, a method for diagnosing an HIV
infection is provided, the method comprising detecting a lymphocyte
or leucocyte infected with the HIV virus using an eDAR method
and/or apparatus provided herein.
[0148] In yet another embodiment, a method for diagnosing a disease
associated with a prion is provided, the method comprising
detecting a prior in a biological fluid from a human or other
animal (e.g., a cow) using an eDAR method and/or apparatus provided
herein. In one embodiment, the disease associated with a prion is
mad cow disease.
[0149] 1. Diagnosing Cancer
[0150] In one particular embodiment, the method comprises detecting
a circulating tumor cell in a blood sample from a subject using an
eDAR method and/or apparatus provided herein. In certain
embodiments, the subject may be a patient who has previously been
diagnosed with Stage I, Stage II, Stage III, or Stage IV cancer. In
certain embodiments, wherein a CTC is detected in a blood sample
from a patient previously diagnosed with cancer, the patient may be
further diagnosed with metastatic cancer.
[0151] In one embodiment, a method is provided for diagnosing
metastatic cancer in a subject that has previously been diagnosed
with a solid tumor, the method comprising the steps of: (a)
detecting the presence or absence of a CTC in an aliquot of a blood
sample from the subject; (b) assigning a value to the aliquot based
on the presence or absence of the CTC; and (c) directing the flow
or collection of the aliquot based on the assigned value. In one
embodiment, the absence of CTCs in the blood sample is correlated
with the subject not having metastatic cancer. In another
embodiment, the presence of at least one CTC in the blood sample is
correlated with the subject having metastatic cancer. In yet
another embodiment, the presence of at least a reference number of
CTCs in the blood is correlated with the subject having metastatic
cancer. In some embodiments, the method may further comprise a step
of (d) diagnosing the subject as not having metastatic cancer if no
CTCs are detected in the blood sample or diagnosing the subject as
having metastatic cancer if at least one CTC is detected in the
blood sample.
[0152] In a related embodiment, a method for monitoring a subject
diagnosed with cancer is provided comprising detecting the presence
or absence of a CTC in an aliquot of a blood sample from the
subject using an eDAR method provided herein. In certain
embodiments, the patient may be monitored for the progression of
cancer to metastatic cancer at regular intervals, for example ,at
least once a year, at least twice a year, or at least about 3, 4,
5, 6, 7, 8, 9, 10, or more times a year. In some embodiments, the
subject may be monitored about once a month, or at least about 2,
3, 4, 5, 6, 7, 8, 9, 10, or more times a month. In one embodiment,
the absence of CTCs in the blood sample is correlated with the
subject not having metastatic cancer. In another embodiment, the
presence of at least one CTC in the blood sample is correlated with
the subject having metastatic cancer. In yet another embodiment,
the presence of at least a reference number of CTCs in the blood is
correlated with the subject having metastatic cancer. In some
embodiments, the method may further comprise a step of (d)
diagnosing the subject as not having metastatic cancer if no CTCs
are detected in the blood sample or diagnosing the subject as
having metastatic cancer if at least one CTC is detected in the
blood sample.
[0153] In embodiments wherein a CTC is detected in a blood sample,
the method may further comprise a step of subjecting one or more
aliquots identified as containing a CTC to further analysis to
identify one or more characteristics of the CTC cell or cells. For
example, an aliquot or pool of aliquots containing a CTC may be
contacted with one or more detection reagents specific for one or
more cancer-specific surface antigens. By determining which
cancer-specific antigens are present on the surface of the CTCs,
therapy can then be designed to target the expressed surface
antigen. Non-limiting examples of cancer-specific surface antigens
that can be assayed for include, without limitation, BCR-ABL or
PDGFR (targeted by drug Gleevec), ERBB2 (targeted by Herceptin),
EFGR (targeted by Iressa, Tarceva), RAR-alpha (targeted by ATRA),
Oestrogen receptor (targeted by Tamoxifen), aromatase (targeted by
Letrazole), androgen receptor (targeted by Flutamide, Biclutamide),
CD20 (targeted by Rituximab), VEGF-receptor (targeted by Avastin),
and the like. Accordingly, in certain embodiments, the method may
further comprise a step of assigning a targeted therapy to the
subject based on the detection of a specific surface antigen
present on the CTC.
[0154] In certain embodiments, the further analysis can be
performed using an eDAR method provided herein, for example by
contacting the pooled aliquots with a plurality of differentially
labeled detection reagents under conditions suitable to transform
the detection reagents into complexes with the cancer-specific
antigens present on the surface of the CTCs and detecting the
complexes using a plurality of detection devices.
[0155] In other embodiments, the further analysis can be performed
by coupling the initial eDAR method with a traditional flow
cytometry or immunochemical method (e.g., immunoblot, ELISA, xMAP
multiplex assay, etc.). In certain embodiments, the eDAR device
used to detect the CTC may be in fluid communication with a second
device or means for performing the further analysis.
[0156] In embodiments wherein a subject is diagnosed with
metastatic cancer, the method may further comprise a step of
assigning therapy for metastatic cancer to the subject.
[0157] In another particular embodiment, a method is provided for
diagnosing a subject with cancer, the method comprising detecting a
CTC in a blood sample taken from the subject. For example,
detecting a CTC in a blood sample from a subject that has not been
previously diagnosed with cancer. In some embodiments, the subject
may have an increased risk of having or developing cancer, for
example, the subject may have a family history of cancer, be a
smoker, or otherwise been exposed to a carcinogenic substance
(i.e., asbestos, benzene, cadmium, radon, radioactivity, and the
like).
[0158] As such, in certain embodiments, a method is provided for
monitoring a subject that has not been previously diagnosed with
cancer, the method comprising the steps of: (a) detecting the
presence or absence of a CTC in an aliquot of a blood sample from
the subject; (b) assigning a value to the aliquot based on the
presence or absence of the CTC; and (c) directing the flow or
collection of the aliquot based on the assigned value. In one
embodiment, the absence of CTCs in the blood sample is correlated
with the subject not having metastatic cancer. In another
embodiment, the presence of at least one CTC in the blood sample is
correlated with the subject having cancer. In yet another
embodiment, the presence of at least a reference number of CTCs in
the blood is correlated with the subject having cancer or
metastatic cancer. In some embodiments, the method may further
comprise a step of (d) diagnosing the subject as not having cancer
if no CTCs are detected in the blood sample or diagnosing the
subject as having cancer if at least one CTC is detected in the
blood sample.
[0159] In certain embodiments, the step of detecting the presence
or absence of the CTC can be performed as described above, for
example, by (i) contacting a biological fluid from the subject with
a detection reagent under conditions suitable to transform the
detection reagent into a complex comprising said detection reagent
and a rare particle; and (ii) detecting the presence or absence of
a complex formed in step (i) in an aliquot of the biological fluid,
or by (i) interrogating the aliquot with an external source of
electromagnetic radiation; and (ii) detecting fluorescence of the
rare particle.
[0160] 2. Methods for Providing a Prognosis
[0161] In one aspect, the present invention provides methods for
providing a prognosis for a disease or condition associated with
the presence of a rare particle in a biological fluid. In one
embodiment, the method comprises the steps of: (a) detecting the
presence or absence of the rare particle in an aliquot of a
biological sample from a subject; (b) assigning a value to the
aliquot based on the presence or absence of the rare particle; (c)
directing the flow or collection of the aliquot based on the
assigned value; and (d) providing either a good prognosis if no
rare particles are detected in the sample or a poor prognosis if a
rare particle is detected in the sample.
[0162] In other embodiments, the aliquot is assigned a value based
on the quantity or the identity of the rare particle in the
aliquot. In certain of these embodiments, a good or poor prognosis
is provided based on the quantity of the rare particles in the
sample. For example, in one embodiment, a good prognosis is
provided if the quantity of the rare particles in the sample is
less than a predetermined reference value and a poor prognosis is
provided if the quantity of the rare particles in the sample is
equal to or greater than the reference value.
[0163] In certain embodiments, a predetermined reference value may
be associated with a likelihood of responding to a particular
therapy or a likelihood of overall or disease free survival for a
period of time, for example at least 6 month, or at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, or more years.
[0164] In certain embodiments, the step of detecting the presence
or absence of the rare particle can be performed as described
above, for example, by (i) contacting a biological fluid from the
subject with a detection reagent under conditions suitable to
transform the detection reagent into a complex comprising said
detection reagent and a rare particle; and (ii) detecting the
presence or absence of a complex formed in step (i) in an aliquot
of the biological fluid, or by (i) interrogating the aliquot with
an external source of electromagnetic radiation; and (ii) detecting
fluorescence of the rare particle.
[0165] In certain embodiments, a method provided herein may be used
to provide a prognosis for any disease associated with a rare
particle. In one embodiment, a method for providing a prognosis for
malaria is provided, the method comprising determining the number
of erythrocytes infected with Plasmodium in a blood sample from an
individual using an eDAR method and/or apparatus provided herein
and providing either a good prognosis if the total number of
infected erythrocytes detected in the blood sample is less than a
predetermined reference value or a poor prognosis if the total
number of infected erythrocytes detected in the sample is equal to
or greater than the reference value.
[0166] In another embodiment, a method for providing a prognosis
for an HIV infection is provided, the method comprising determining
the number of lymphocytes or leucocytes infected with an HIV virus
in a blood sample from an individual using an eDAR method and/or
apparatus provided herein and providing either a good prognosis if
the total number of infected cells detected in the blood sample is
less than a predetermined reference value or a poor prognosis if
the total number of infected cells detected in the sample is equal
to or greater than the reference value.
[0167] In yet another embodiment, a method for providing a
prognosis for a disease associated with a prion is provided, the
method comprising determining the number of prions in a biological
fluid sample from a subject using an eDAR method and/or apparatus
provided herein and providing either a good prognosis if the total
number of prions detected in the sample is less than a
predetermined reference value or a poor prognosis if the total
number of prions detected in the sample is equal to or greater than
the reference value.
[0168] In certain embodiments, a predetermined reference value may
be associated with a likelihood of responding to a particular
therapy or a likelihood of overall or disease free survival for a
period of time, for example at least 6 month, or at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, or more years.
[0169] In a specific embodiment, the present invention provides a
method for providing a prognosis for a subject diagnosed with a
solid tumor is provided. In one embodiment, the method comprises
the steps of (a) detecting the presence or absence of a CTC in an
aliquot of a blood sample from the subject; (b) assigning a value
to the aliquot based on the presence or absence of the CTC; (c)
directing the flow or collection of the aliquot based on the
assigned value; and (d) providing either a good prognosis if no
CTCs are detected or a poor prognosis if a CTC is detected.
[0170] In another embodiment, a method is provided for providing a
prognosis for a subject diagnosed with metastatic cancer, the
method comprising the steps of (a) detecting the presence or
absence of a CTC in an aliquot of a blood sample from the subject;
(b) assigning a value to the aliquot based on the number CTCs
detected in the aliquot; (c) directing the flow or collection of
the aliquot based on the assigned value; and (d) providing either a
good prognosis if the total number of CTCs detected in the blood
sample is less than a predetermined reference value or a poor
prognosis if the total number of CTCs detected in the sample is
equal to or greater than the reference value.
[0171] In certain embodiments, a predetermined reference value may
be associated with a likelihood of responding to a particular
therapy or a likelihood of overall or disease free survival for a
period of time, for example at least 6 month, or at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, or more years.
[0172] 3. Monitoring Disease Progression or Response to Therapy
[0173] In another aspect, the present invention provides methods
for monitoring the progression of a disease or the response to a
therapy, the method comprising detecting a rare particle in a fluid
sample using an eDAR method and/or apparatus provided herein.
[0174] In one embodiment, the method comprises the steps of: (a)
detecting the presence or absence of the rare particle in a
plurality of aliquots of a first biological sample taken from a
subject at a first time; (b) assigning a value to the aliquots
based on the presence, absence, quantity, or identity of the rare
particle; (c) determining the total value of all the aliquots from
the first sample; (d) detecting the presence or absence of the rare
particle in a plurality of aliquots of a second biological sample
taken from the subject at a second time; (e) assigning a value to
the aliquots based on the presence, absence, quantity, or identity
of the rare particle; (f) determining the total value of all the
aliquots from the second sample; and (g) comparing the total value
assigned to the first sample to the total value assigned to the
second sample, wherein an increased value assigned to the second
sample as compared to the first sample is correlated with a
progression of the disease and/or a poor response to the therapy
and/or a decreased value assigned to the second sample as compared
to the first sample is correlated with a regression of the disease
and/or a good response to the therapy.
[0175] In certain embodiments, the aliquots may further be directed
into a particular channel or chamber (channeled) based on the value
assigned for collection, further enrichment, or further
analysis.
[0176] In certain embodiments, methods of monitoring disease
progression or response to therapy may be employed on a regular
basis after diagnosis of the disease or initiation of the treatment
regime. For example, samples may be collected from a subject at
least once a year, at least twice a year, or at least about 3, 4,
5, 6, 7, 8, 9, 10, or more times a year. In some embodiments, the
subject may be monitored about once a month, or at least about 2,
3, 4, 5, 6, 7, 8, 9, 10, or more times a month.
[0177] In certain embodiments, wherein a progression of the disease
or poor response to a therapy is found, the method may further
comprise a step of assigning a therapy, increasing a dosage regime,
changing a therapeutic regime, and the like.
[0178] In certain embodiments, the disease or condition associated
with a rare particle may be cancer, malaria, HIV/Aids, a
prion-related disease, or the like.
[0179] C. Methods of Monitoring Water Quality
[0180] In another aspect, the present invention provides a method
for monitoring water quality by detecting one or more rare particle
contaminant in a sample of water using an eDAR method or apparatus
provided herein.
[0181] In one embodiment, the method comprises the steps of (a)
detecting the presence or absence of a water contaminant in an
aliquot of a sample taken from a water source; (b) assigning a
value to the aliquot based on the presence, absence, quantity, or
identity of the water contaminant in the aliquot; and (c) directing
the flow or collection of the aliquot based on the assigned value,
whereby the quality of the water source is determined based on the
total value assigned to all of the aliquots detected in the
method.
[0182] In certain embodiments, the water source may be a lake,
pool, river, stream, or other natural body of water. In certain of
these embodiments, the water may be tested to determine or predict
the impact a man made object or activity has or will have on the
body of water or to assess the feasibility or safety of using the
body of water to supply drinking water to a population.
[0183] In other embodiments, the water source may be a pool or pond
at a water treatment plant, a reservoir, a water tower, or other
body of water collected for the purpose of supplying drinking water
to a population. In certain of these embodiments, the water may be
tested to assess the feasibility or safety of using the body of
water to supply drinking water to a population.
[0184] In certain embodiments a method provided herein may be used
to regularly monitor the quality of a water source used to supply
drinking water to a population, for example at a water treatment
plant or in a water tower or reservoir. In such an embodiment, the
water source may be monitored at least once a year, at least twice
a year, or at least about 3, 4, 5, 6, 7, 8, 9, 10, or more times a
year. In some embodiments, the subject may be monitored about once
a month, or at least about 2, 3, 4, 5, or more times a month. In
yet other embodiments, the water may be tested at least once a
week, or at least 2, 3, 4, 5, 6, or more times a week, or at least
daily or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times a
day.
[0185] In other embodiment, a method provided herein may be used to
determine the feasibility or safety of using a body of water to
supply drinking water to a population after a natural disaster
(e.g., after a hurricane, tsunami, or earthquake), accident, or act
of terrorism.
[0186] Methods of testing or monitoring water safety or quality may
comprise the detection of a rare particle that is a water
contaminant, for example a parasite such as a species of Giardia,
Cryptosporidium, or other organic or inorganic water contaminant
that when present at low quantities poses a public health risk.
[0187] D. Apparatuses
[0188] In one aspect, the present invention provides a device for
detecting a rare particle in a biological fluid.
[0189] In one embodiment, the device comprises: (a) at least a
first input channel; (b) at least two exit channels; (c) at least
one detector capable of detecting one or more rare particles in an
aliquot of the biological fluid; (d) a mechanism for directing the
flow of the aliquot; and (e) a ranking device capable of assigning
a value to the aliquot based on the presence, absence, identity,
composition, or quantity of the rare particles in the aliquot,
wherein the computer is in communication with the detector and the
mechanism for directing the flow of the aliquot.
[0190] In some embodiments, the apparatus further comprises a
source for interrogating the aliquot. In other embodiments, wherein
the rare particle or cell intrinsically exhibits chemiluminescence
or bioluminescence, the apparatus may not require a source for
interrogating the aliquot.
[0191] In certain embodiments, the apparatus provided herein may
comprise a flow channel enclosed by walls and/or microfabricated on
a substrate, with design features to minimize inadvertent damage to
rare cells. Reducing inadvertent damage of rare cells reduces the
rate of false-negative which could lead to erroneous patient
diagnosis or prognosis. The flow channel may further comprise
channels with hydrodynamically designed apertures to exclude
biological cells with minimal stress or damage as described in US
Patent Application Nos. 2007/0037172 and 2008/0248499. Such
channels, referred to in the aforementioned patent applications as
channels with one-dimensional ("1-D") apertures, reduce the
hydrodynamic pressure experienced by the cells during the cell
exclusion process and therefore reduce the likelihood of cell
lysis. Channels with 1-D apertures may be strategically arranged in
an array according to "effusive filtration" configuration as
described in US Patent No. 2008/0318324 to further re-direct,
partition, dampen, or disperse the flow, consequently reducing the
force of impact experienced by the cells at the moment of
exclusion. The walls that enclose the flow channel may be
fabricated using a UV-curing process in accordance with the
procedures described in PCTPCT/US2009/02426, from a biocompatible
substrate material that is a medical-device grade polymer, so that
the eDAR apparatus would be in compliance with regulations
governing medical device manufacturing.
[0192] 1. Mechanisms for Directing the Flow of an Aliquot
[0193] In certain embodiments, the mechanism for directing the flow
directs the flow of the aliquot into either a first exit channel if
the aliquot contains a rare particle or a second exit channel if
the aliquot does not contain a rare particle.
[0194] In another embodiment, the mechanism for directing the flow
directs the flow of an aliquot containing a rare particle into one
of a plurality of exit channels depending on the identity,
composition, or quantity of the rare particle.
[0195] In certain embodiments, the mechanism for directing the flow
of the aliquot comprises an electrode, a magnetic element, an
acoustic element, an electro-actuated element, an electric field,
or a magnetic field.
[0196] In yet other embodiments, the mechanism for directing the
flow of the aliquot comprises one or more electro-actuated valves
or pistons, wherein the valves or pistons control the flow of a
liquid in at least a first directional flow channel that intersects
with the first input channel and the two exit channels at a first
junction.
[0197] In one embodiment, solenoid pistons are subcomponents of
electro-actuated solenoid valves. In another embodiment, solenoid
pistons are embedded in device by molding. In yet another
embodiment, the embedded solenoid pistons may be replaced by
solenoid valves in fluidic communication via tubings.
[0198] In one particular embodiment, an apparatus provided herein
may comprise one or more electrodes for tracking and/or
manipulating the trajectory or flow of a particle, aliquot, or
fluid sample. In certain embodiments, the electrode may enhance the
separation of an aliquot based on phenomena such dielectrophoresis
or electrowetting.
[0199] In certain embodiments, the apparatuses of the present
invention may comprise one or more acoustical elements for tracking
and/or manipulating the trajectory or flow of a particle, aliquot,
or fluid sample. In certain embodiments, acoustical elements may be
used to manipulate the trajectory of select particles or cells with
acoustical energy (e.g., acoustophoresis, ultrasonic or megasonic
waves) to improve cell separation based on the response of cells to
compressive pressure waves.
[0200] In another embodiment, the apparatuses provided herein may
further comprise a magnetic element for the separation of a rare
particle or cell bound to or bound by a magnetic particle. In
certain embodiments, the magnetic element may enhance the
separation of an aliquot, particle, or cell based on the magnetic
susceptibility of the cells or the micro-magnetic or nano-magnetic
particles attached to a particle or cell.
[0201] In certain embodiments, an apparatus provided herein may
comprises the use of fluidic pressure changes, flow-rate changes,
or electroosmostic flow changes to manipulate the trajectory of
select particles or cells.
[0202] 2. Detection Devices
[0203] In certain embodiments, the detector is selected from the
group consisting of a camera, an electron multiplier, a
charge-coupled device (CCD) image sensor, a photomultiplier tube
(PMT), an avalanche photodiode (APD), a single-photon avalanche
diode (SPAD), and a complementary metal oxide semiconductor (CMOS)
image sensor.
[0204] In certain embodiments, an apparatus provided herein may
comprise a photo, electro, acoustical or magnetic detector to track
the motion of select cells or to enumerate select particles or
cells present in an aliquot.
[0205] In some embodiments, an apparatus or method provided herein
may incorporate fluorescence (single or multi-color) microscopy
imaging in various configurations, which include but are not
limited to bright-field, epi, confocal, DIC (differential
interference contrast), dark-field, Hoffman, or phase-contrast.
[0206] In some embodiments, the apparatuses provided herein may
comprise a plurality of detection devices, for example, at least 2,
3, 4, 5, 6, 7, 8, 9, 10, or more detection devices. Multiple
detection devices may be necessary for performing a methods of the
present invention, for example, wherein more than one rare particle
or cell is present in a fluid sample, more than one cell marker is
being used to differentiate different cell types, or multiple
detection reagents are being detected simultaneously.
[0207] 3. Interrogation Devices
[0208] In certain embodiments, the apparatuses provided herein may
further comprise a source for interrogating or exciting a
detectable moiety present in an aliquot. In certain embodiments,
the source for interrogating is selected from, for example, a laser
(solid state, diode-pumped, ion, or dye), a light-emitting diode
(LED), a lamp, an arc discharge, a magnetic pulse, or a natural
light source.
[0209] In some embodiments, the apparatuses provided herein may
comprise a plurality of interrogation devices, for example, at
least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detection devices.
Multiple interrogation devices may be necessary for performing a
methods of the present invention, for example, wherein more than
one rare particle or cell is present in a fluid sample, more than
one cell marker is being used to differentiate different cell
types, or multiple detection reagents are being detected
simultaneously.
[0210] 4. Ranking Devices
[0211] In certain embodiments, a ranking device may be selected
from a computer, a controller, a chip with integrated circuits, a
circuit board, an electronic element, software, an algorithm, or a
combination thereof.
[0212] 5. Flow Channels and Chambers
[0213] In certain embodiments, an apparatus provided herein may
comprise a plurality of flow channels, including one or more input
flow channels (i.e., channels that bring an aliquot to a detection
volume) and one or more output channels (i.e., channels that take
an aliquot away from a detection volume. In some embodiments, an
apparatus as provided herein may comprise a combination of at least
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more
input channels and at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, or more output channels.
[0214] In certain embodiments, an apparatus may comprise multiple
flow channels connecting to the main channel to inject additional
fluid to alter the local velocity.
[0215] In one embodiment, an apparatus provided herein may comprise
a flow channel or chamber enclosed by walls fabricated from
materials including, but not limited to, polymeric materials
(polydimethylsiloxane (PDMS), polyurethane-methacrylate (PUMA),
polymethylmethacrylate (PMMA), polyethylene, polyester (PET),
polytetrafluoroethylene (PTFE), polycarbonate, parylene, polyvinyl
chloride, fluoroethylpropylene, lexan, polystyrene, cyclic olefin
copolymers, polyurethane, polyestercarbonate, polypropylene,
polybutylene, polyacrylate, polycaprolactone, polyketone,
polyphthalamide, cellulose acetate, polyacrylonitrile, polysulfone,
epoxy polymers, thermoplastics, fluoropolymer, and polyvinylidene
fluoride, polyamide, polyimide), inorganic materials (glass,
quartz, silicon, GaAs, silicon nitride), fused silica, ceramic,
glass (organic), metals and/or other materials and combinations
thereof.
[0216] In certain embodiments, a wall materials can be fabricated
of porous membranes, woven or non-woven fibers (such as cloth or
mesh) of wool, metal (e.g. stainless steel or Monel), glass, paper,
or synthetic (e.g. nylon, polypropylene, polycarbonate, parylene,
and various polyesters), sintered stainless steel and other metals,
and porous inorganic materials such as alumina, silica or
carbon.
[0217] In certain embodiments, the apparatuses provided herein may
comprise a flow channel or chamber that has been pre-treated with a
chemical or biological molecule. For example, a channel or chamber
may be treated with an anticoagulant compound to prevent or reduce
the association of a particle in the fluid sample, a compound that
preferentially binds to a particle in the fluid sample, for example
a rare particle or cell, or a compound that prevents or reduces the
agglomeration or aggregation of a particle in the fluid sample.
[0218] In one embodiment, the channel or chamber surfaces may be
treated with anticoagulant compounds, compounds that preferentially
bind to circulating tumor cells, or compounds that prevent the
sticking of cells.
[0219] In certain embodiments, a channel or chamber surface may be
modified chemically to enhance wetting or to assist in the
adsorption of select cells, particles, or molecules.
Surface-modification chemicals may include but not limited to
silanes such as trimethylchlorosilane (TMCS), hexamethyldisilazane
(HMDS), (Tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane,
chlorodimethyloctylsilane, Octadecyltrichlorosilane (OTS) or
.gamma.-methyacryloxypropyltrimethyoxy-silane; polymers such as
acrylic acid, acrylamide, dimethylacrylamide (DMA), 2-hydroxyethyl
acrylate, polyvinylalcohol (PVA), poly(vinylpyrrolidone (PVP),
poly(ethylene imine) (PEI), Polyethylene glycol (PEG), epoxy
poly(dimethylacrylamide (EPDMA), or PEG-monomethoxyl acrylate;
surfactants such as Pluronic surfactants, Poly(ethylene
glycol)-based (PEG) surfactants, sodium dodecylsulfate (SDS)
dodecyltrimethylammonium chloride (DTAC), cetyltriethylammonium
bromide (CTAB), or Polybrene (PB); cellulose derivatives such as
hydroxypropylcellulose (HPC), or hydroxypropylmethylcellulose
(HPMC); amines such as ethylamine, diethylamine, triethylamine, or
triethanolamine, fluorine-containing compounds such as those
containing polytetrafluoroethylene (PTFE) or Teflon.
[0220] 6. Means for Background Reduction
[0221] In certain embodiment, the apparatuses provided herein may
further comprise a means for reducing excessive background signal
and/or improving the signal-to-noise ratio. By reducing excessive
background signal and increasing the signal-to-noise ratio, the
sensitivity of detection is enhanced as the weak signals from even
a highly diluted aliquot can be accurately detected. In other
words, the better the signal-to-noise ratio, larger an aliquot can
be scanned. As a direct result, the fluidic throughput is
correspondingly increased since fewer (but larger) aliquots need to
be scanned.
[0222] In one embodiment, the means for reducing background signal
comprises a mask. A mask can consist of any number of apertures of
any shape or size, positioned in any orientation with or without
any periodic spacing. For example, FIG. 3 illustrates a mask (301)
containing an array of apertures (311), positioned between the
detection volume and a detector to selectively allow through a
detectable characteristic.
[0223] In certain embodiments, a mask may comprise an optical
element that selectively pass through certain wavelengths of light,
for example, a low-pass, high-pass, or band-pass filter, or
acousto-optic modulator, spatial-light modulator, light-chopper,
fabricated hologram, physical aperture, or galvo scanner.
[0224] In other embodiments, a mask can consist of magnetic
elements that selectively prevent passage of a magnetic field.
[0225] In some embodiments, other devices that accomplish similar
gains in signal-to-noise ratio may be used in place of or in
conjunction with a mask. For example, in certain embodiments a
device selected from a lock-in amplifier, a scanning detector, a
modulated interrogator or detector, or any apparatuses that
modulate frequency or intensity may be used to increase the
signal-to-noise ratio.
[0226] In yet other embodiments, detectors with spatial-modulation
functionality of a mask directly incorporated within may be used in
conjunction with the apparatuses provided herein. In certain
embodiments, a separate mask is not present. In other embodiments,
a separate mask is also present. Non-limiting examples of detectors
with incorporated mask functionality include photodiode arrays or
cameras with spatial pixelation such that signals of individual
photodiodes or select pixels of cameras may be removed or kept.
[0227] 7. Additional Elements
[0228] In certain embodiments, the apparatuses provided herein may
further comprise additional elements useful for performing assays,
processes, or tests in a fashion that is coupled to the eDAR
methods provided herein.
[0229] In one embodiment, an apparatus provided herein may further
comprise one or more resistive heating elements to perform on-chip
cellular assays such as Polymerase Chain Reaction (PCR) or
Real-Time Polymerase Chain Reaction (RT-PCR).
[0230] In yet other embodiments, an apparatus provided herein may
further comprises one or more electrodes, for example, to conduct
on-chip chemical assay such as electrophoresis or
eletrochromatography.
[0231] In another embodiment, an apparatus provided herein may
further comprise a filter element. In a particular embodiment, the
filter element may be in the form of microposts, microimpactors,
microsieves, channels with apertures smaller than bioparticles,
channels with apertures such that a bioparticle is prevented from
entering an apertured but fluid is allowed to continue to flow
around the bioparticle through the aperture ("1-D channels"),
microbeads, porous membranes, protrusions from the walls, adhesive
coating, woven or non-woven fibers (such as cloth or mesh) of wool,
metal (e.g. stainless steel or Monel), glass, paper, or synthetic
(e.g. nylon, polypropylene, polycarbonate, parylene, and
polyester), sintered stainless steel or other metals, or porous
inorganic materials such as alumina, silica, or carbon.
[0232] For example, FIG. 16 illustrates a filter element (1622),
which may be disposed in a channel, such as an outlet channel
(1621), to selectively allow the passage of fluid portion while
retaining the desired bioparticles.
[0233] In yet another embodiment, an apparatus provided herein may
be coupled to a conventional flow cytometer.
[0234] For example, FIG. 16 illustrates an outlet channel (1621)
that may be in fluidic communication to a conventional flow
cytometer (with or without filter element 1622) such that discrete
aliquot 1661 containing rare cell 1602 is further examined or
sorted serially (one cell by one cell).
[0235] IV. EXAMPLES
[0236] A. Example 1
[0237] An example of an eDAR apparatus with a single inlet and
outlet port for the detection or quantitation of rare particles in
a fluid.
[0238] FIG. 4 shows an example of eDAR apparatus consisting of a
device (411) to aliquot a cell suspension, an interrogation device
(421 and 422), a detection or imaging device (431, 432, and 433),
and a ranking device (computer not shown). In this particular
example, a device (411) to aliquot a cell suspension may consist of
a fluidic channel (414) contained within walls and fluidic ports
(412 and 413). A laser serves as an interrogation device. An
inverted microscope with photodiodes, photomultipliers, or cameras
is used as a detection device. A mask (451) is placed in a path
between the channel (414) and the detection devices (431, 432, and
433). A computer accepts the signal from the detection device and
through an algorithm ranks the aliquot. The computer then directs
the aliquot into the proper channel based on the value of the
ranking (i.e., the presence, absence, quantity, identity, or
composition of rare particles in the fluid sample). Although FIG. 4
illustrates three detection devices (431, 432, and 433) and two
interrogation devices (421 and 422), in practice eDAR may consist
of only one detection device and one interrogation device, or
multitudes of detection devices and interrogation devices.
[0239] In one use of the apparatus illustrated in FIG. 4, the
interrogation devices (421 and 422) consisted of a 488 nm
solid-state diode pumped laser and a 633 nm HeNe laser which are
directed into an inverted microscope. The two laser beams were
shaped using cylindrical optics to form a collimated elliptical
beam with an aspect ratio of 10 to 1 prior to entering the
microscope objective. Using a combination of half-waveplate and
polarizing beam splitter, the intensity of each beam could be
adjusted, while mirrors independently steered the light to create a
spatially co-localized excitation region. The fluorescence from
bioparticles was split into three wavelength bands by two dichroic
mirrors before passing through the bandpass filters and refocused
onto the three single-photon avalanche diodes (SPADs; 431, 432, and
433). One SPAD collected fluorescence in the wavelength range of
560-610 nm, a second SPAD collected fluorescence in the range of
645-700 nm, and a third SPAD collected in the range of 500-540 nm.
The SPAD outputs were directed to a computer with a counter/timer
board and analyzed with several algorithms.
[0240] B. Example 2
[0241] eDAR apparatus with two outlet ports for the detection or
quantitation of rare particles in a fluid.
[0242] FIG. 5 illustrates an eDAR apparatus consisting of a device
to aliquot cell suspension (510), an interrogation device (521), a
detection or imaging device (531), a ranking device (computer not
shown), and an apparatus to direct aliquot according to the
assigned ranking This apparatus also includes a single inlet port
(511), two outlet ports (512 and 513), and three fluidic channels
(514) joined at a single point to aliquot the fluid. A laser serves
as the interrogation device (521) and an inverted microscope with
photodiodes, photomultipliers, or cameras serves as detection
device (531). A mask (551) is placed between the channels (514) and
the detection device (531). A computer accepts the signal from the
detection device and ranks the aliquot through an algorithm. The
device then utilizes an electrical, magnetic, hydrodynamic, or
pneumatic mean to direct the aliquot into either outlet 512 or
outlet 513 according to the assigned ranking
[0243] In addition to the eDAR apparatus described above, which has
a single interrogation device and a single detection devise,
multiple interrogation and detection devices may be used in
conjunction with the eDAR apparatuses described herein. For
example, FIG. 6 illustrates an eDAR apparatus with one
interrogation device (640) and three detection devices (650, 651,
and 652).
[0244] C. Example 3
[0245] eDAR apparatus with multiple inlet and/or outlet ports for
the detection or quantitation of rare particles in a fluid.
[0246] In various aspects of the invention, an eDAR apparatus may
consist of multiple inlet and/or outlet ports. For example, FIG. 7
illustrates a device (710) to aliquot suspension with five ports
(711, 712, 713, 714, and 715) and five fluidic channels 716 joined
at a single point. One or more ports may be used as fluidic inlet;
one or more ports may be used as fluidic outlet. For example, the
apparatus may be used such that two ports are used as inlet ports
and three ports are used as outlet ports, or it may be used such
that one port is an inlet port and four ports are outlet ports,
etc. In theory, the device (710) may contain any number of fluidic
inlets and outlets and any number of fluidic channels. These
fluidic channels may be joined at more than one point and the
joining point need not be circumscribed by the fluidic inlets or
outlets.
[0247] D. Example 4
[0248] Detection of circulating tumor cells (CTCs) in the blood of
breast cancer patients by the use of eDAR.
[0249] Freshly venipunctured blood from Stage IV metastatic breast
cancer patients was drawn from a single puncture into three
separate tubes. The first blood portion (first tube) was discarded
to avoid possible contamination of epithelial cells from skin
puncture. A second portion was collected into Veridex's CellSave
tube containing stabilizing reagents for circulating tumor cell
detection using a Veridex's CellSearch system. A third portion was
collected into a collection tube containing EDTA anticoagulant for
separate analysis using eDAR.
[0250] The third portion was incubated with enzymes, fixatives,
permeability reagents, and fluorescent antibodies targeting
pan-cytokeratin, CD45, and Epithelial Cell Adhesion Molecule
(EpCAM). A positive identification of circulating tumor cell is
defined as a cell expressing pan-cytokeratin and EpCAM, but not
CD45. CD45 is commonly known as leucocyte common antigen and is
indicative of a white blood cell. An object bound with all three
antibodies is deemed false-positive, frequently a result of protein
aggregation.
[0251] Briefly, the 5 to 10 mL antibody-labeled blood samples were
flowed through an eDAR apparatus as described in Example 1 at a
flow rate of between about 10-500 .mu.L/min, using a compressed air
source supplying 7.6 psi to drive the flow. The eDAR apparatus was
operating in the continuous flow (simultaneous) mode. For these
experiments, microchannels that were 200 .mu.m wide by 50 .mu.m
tall were used. For interrogation of the labeled antibody
complexes, line-confocal excitation beams were provided at both 488
nm and 633 nm, which illuminated a sheet of light that was about
5-10 .mu.m thin. As such, the line-confocal detection volume had
dimensions of about 200 .mu.m (width).times.50 .mu.m
(height).times.10 .mu.m (thickness), providing a detection volume
of about 0.1 nL. Three SPAD detection devices were operating at
10,000 Hz sampling rate, configured to detect fluorescence signals
emanating from the cells at 450-610 nm, 645-700 nm, and 500-540 nm.
At this rate, each aliquot was on the order of 1 nL to 50 nL,
estimated to contain 5-250 white blood cells and 5,000-25,000 red
blood cells. For a sample of 5-10 mL, it thus takes between 10-20
min to process the sample at a flow rate of 500 .mu.L/min. A 5 mL
sample processed in this fashion will be divided into 10,000
aliquots having a volume of 50 nL each.
[0252] As such, if an eDAR apparatus having two outlet channels was
used, a 5 mL sample containing 200 CTCs can be reduced to a volume
of 10 .mu.L in only 10 minutes, without the use of a filter. If the
eDAR apparatus was further in liquid contact with a flow cytometer,
All of the 200 CTC cells present in the 5 mL sample could be
counted and/or individually isolated in only 2% of the normal time
by implementing an eDAR step prior to the flow cytometry.
[0253] FIG. 8 shows CTC counts from 27 blood samples from Stage IV
breast cancer patients (of multiple patients drawn on different
dates) using Veridex's CellSearch system (lower panel) and eDAR
(upper panel). As can be readily noted, most patient samples
registered zero CTC counts using CellSearch system. In contrast,
more than 50% of the same patient blood draws analyzed by eDAR were
found to contain 200-400 CTC counts. This amply demonstrates a
hundred times more sensitivity with the use of eDAR, as compared to
the use of Veridex's commercial CellSearch system. The heightened
sensitivity of eDAR is attributable to accurate discrimination
between CTCs and background by aliquot ranking and the use of a
mask.
[0254] As also shown in FIG. 8, 100% of the patient samples
analyzed by eDAR indicated the presence of CTCs, whereas only 40%
of the patient samples analyzed by Veridex's CellSearch system
indicated any presence of any CTCs. This demonstrated a
significantly lower rate of false-negative provided by eDAR. High
rate of false-negative in a CTC test can lead to an inaccurate
prognosis of metastasis and give patients a false sense of security
that the existing treatment regimen is sufficient.
[0255] Exemplary images of a CTC detected by this eDAR method are
provided in FIG. 9. Briefly, this figure shows the optical images
of cancer cells trapped from patient blood using eDAR (arrows mark
CTCs) under various illumination. FIG. 9A is a brightfield image of
a CTC amidst red blood cells. FIG. 9B is a fluorescence image
indicating the presence of pan-cytokeratin. FIG. 9C is a
fluorescence image indicating the absence of CD45, hence ruling out
the possibility of false-identifying a white blood cell as a
CTC.
[0256] E. Example 5
[0257] Detection of cancer stem cells among a population of cancer
cells.
[0258] Cancer cells segregated by eDAR may be further analyzed to
distinguish the subpopulations within the biological fluid. By
perfusing with additional fluorescent antibodies targeting specific
proteins, some cancer cells may be distinguished from others. For
example, cancer cells that express CD44 but not CD24 proteins have
recently been called cancer stem cells for their association with
high metastatic potential. Other proteins may be associated with
various traits of cells.
[0259] For example, FIG. 10 demonstrates the identification of
breast cancer stem cells (marked with arrowheads). Briefly, breast
cancer cells (MCF-7) were labeled with Alexa 488-anti-CD44
(positive) and Alexa 647-anti-CD24 (negative). FIG. 10A is a
fluorescence image (500-540 nm) for detecting Alexa 488-anti-CD44
(green); FIG. 10B is a fluorescence image (645-700 nm) for
detecting Alexa 647-anti-CD24 (red); FIG. 10C is the brightfield
image. FIG. 10D is a composite image indicating CD44+/CD24-(arrows
indicate cancer stem cells). Approximately 25% of cancer cells were
found to express CD44 but not CD24 and thus meet the criteria for
cancer stem cells. In this fashion, eDAR may be used to distinguish
sub-populations of cancer cells in a biological fluid.
[0260] F. Example 6
[0261] eDAR Detection Using Discrete Aliquots.
[0262] In one example of the methods provided herein, eDAR may
operated by using discrete aqueous aliquots that are separated by
an immiscible phase to encapsulate bioparticles prior to the
detection step. FIG. 16 illustrates an eDAR apparatus operating in
this fashion. For example, a cell suspension containing undesired
cells (1601) and desired rare cells 1602 is partitioned into
discrete aliquots (1631, 1641, 1651, and 1661), which are separated
from one another by an immiscible phase (1642). The discrete
aliquots are directed to flow from let to right in a flow channel
(1603). As aliquot 1641 traverses the detection volume (1604;
cylindrical outline), multiple cells encapsulated within the
discrete aliquot (1641) are detected simultaneously. If no desired
cells are detected, the discrete aliquot (1641) is ranked as null
and directed toward Channel 1611 (see, for example, aliquot 1651).
If any desired rare cells are detected, the discrete aliquot is
ranked as nonzero and is directed toward Channel 1621 (see, for
example, aliquot 1661).
[0263] In one embodiment, filter element 1622 may be disposed in
channel 1621 to selectively allow the passage of fluid portion
while retaining the desired bioparticles. In one embodiment, and
eDAR apparatus may be coupled to a conventional flow cytometer. For
example, in FIG. 16, channel 1621 may be in fluidic communication
to a conventional flow cytometer (with or without filter element
1622), such that discrete aliquot 1661 containing rare cell 1602 is
further examined or sorted serially (one cell at a time).
[0264] The immiscible phase (1642) may be continuous (i.e.,
surrounds the discrete aliquots entirely) as illustrated in FIG. 16
or segmented (i.e., immiscible phase 1642 occupies only the spacing
between discrete aliquots but does not completely surround the
aliquots).
[0265] G. Example 7
[0266] As an example of the utility of one aspect of the present
invention, if a 10-mL cell suspension contains only 9 desired rare
cells amidst 10 billion undesired cells, eDAR, in the simplest
form, would require the detection of a characteristic from the
desired cells contained in 10 aliquots. Since there are only 9
desired cells, at least one aliquot would be devoid of the desired
cells and can be ranked as null and discarded immediately. The
undesired cells contained in the discarded portion would not need
to be screened individually. Consequently, with merely 10 aliquots
at least 1/10 of total volumes is immediately discarded and 1/10 of
the undesired cells (contained within the discarded volume) would
not need to be detected individually. To put it in perspective,
that is 1 billion undesired cells eliminated as an ensemble with
one decision. With current state-of-the-art cell sorter operating
at the extreme speed of 70,000 objects/sec, this one decision
resulted in 1,000,000,000/70,000=14,300 sec or 4 hours of time
saved. This results in a significant increase in time
efficiency.
[0267] Following from the scenario presented above, suppose if the
10-mL cell suspension is partitioned into 100 aliquots of 100 .mu.L
each, since the entire volume of cell suspension contains only 9
desired cells, at least 91 portions would not contain any desired
cells. Therefore by performing only 100 scans and make 100
decisions, 91 aliquot.times.100 .mu.L=9.1 mL can be immediately
eliminated. The cells contained within the discarded portions would
be 9.1 billion cells, or 91% of the undesired cells are eliminated
within 100 decisions.
[0268] H. Example 8
[0269] FIG. 1 illustrates a particular embodiment of the invention,
wherein a rare particle characteristic is detected in an aliquot
during operation of a simultaneous mode. Briefly, a cell suspension
containing undesirable cells (101) and desirable rare cells (102)
is directed to flow from left to right in a flow channel (103).
Multiple cells may traverse a detection volume (104) enclosed by a
shaded cylinder at a given time and be detected simultaneously. If
no desired cells are detected, an aliquot equivalent to the
detection volume is ranked as null and directed toward Channel 111.
If any desired cells are detected, the aliquot is ranked as nonzero
and is directed toward Channel 121.
[0270] A filter element (122) may optionally be disposed in Channel
121 to selectively allow the passage of the fluid portion while
retaining the desired bioparticles. The filter element may be in
the form of microposts, microimpactors, microsieves, channels with
apertures smaller than bioparticles, channels with apertures such
that a bioparticle is prevented from entering an aperture but fluid
is allowed to continue to flow around the bioparticle through the
aperture ("1-D channels"), microbeads, porous membranes,
protrusions from the walls, adhesive coating, woven or non-woven
fibers (such as cloth or mesh) of wool, metal (e.g. stainless steel
or Monel), glass, paper, or synthetic (e.g. nylon, polypropylene,
polycarbonate, parylene, and polyester), sintered stainless steel
or other metals, or porous inorganic materials such as alumina,
silica, or carbon.
[0271] I. Example 9
[0272] In another example of eDAR run in a simultaneous mode, the
method may further consist of selectively masking the aliquot as to
reduce excessive background signal and improve the signal-to-noise
ratio. FIG. 2 illustrates the use of a mask (211) with an array of
apertures (212) positioned between the detection volume and a
detector to selectively allow through a detectable characteristic.
Multiple bioparticles still can be simultaneously detected with the
use of the mask (211). By reducing excessive background signal and
increasing the signal-to-noise ratio, the sensitivity of detection
is enhanced as the weak signals from even a highly diluted aliquot
can be accurately detected. In other words, the better the
signal-to-noise ratio, larger an aliquot can be scanned. As a
direct result, the fluidic throughput is correspondingly increased
since fewer (but larger) aliquots need to be scanned. If no desired
bioparticles are detected, an aliquot equivalent to the detection
volume is ranked as null and directed toward Channel 221. If any
desired bioparticles are detected, the aliquot is ranked as nonzero
and is directed toward Channel 222.
[0273] J. Example 10
[0274] FIG. 11 panel A, which is an enlarged illustration of device
710 (FIG. 7), illustrates device 1110, for aliquoting suspension
with five fluidic channels (1111, 1112, 1113, 1114, and 1115)
joined at junction 1116. Fluidic channels 1111, 1112, 1113 carried
fluid toward junction 1116, whereas fluidic channels 1114 and 1115
carried fluid away from junction 1116. Solenoid piston 1120 was
placed on top of channel 1111 and solenoid piston 1121 was placed
on top of channel 1112. Solenoid pistons 1120 and 1121 were
configured to push down on an elastomeric polydimethylsiloxane
(PDMS) membrane, which separated the pistons 1120 and 1121 from the
fluid in the channels 1111 and 1112.
[0275] FIG. 11 panel A illustrates the operation of device 1110 for
aliquoting suspension. By having solenoid piston 1120 pushing down
on the PDMS membrane on top of flow channel 1111, channel 1111 was
closed off. At the same time solenoid piston 1121 was configured to
allow fluid to pass through channel 1112 toward junction 1116. As a
result, blood containing rare cells in the incoming channel 1112
was diverted to left outlet channel 1114 (see panel B, 0 ms). To
direct aliquot 1130 into the right outlet channel 1115, solenoid
piston 1120 was retracted from the PDMS membrane on top of channel
1111 while solenoid piston 1121 pushed down the PDMS membrane on
top of channel 1112. This resulted in directing an aliquot 1130 of
blood containing rare cells into the right outlet channel 1115
(panel C, 5 ms and panel D, 10 ms). Aliquot redirecting using
solenoid pistons as described required as little time as 5 ms.
[0276] K. Example 11
[0277] FIG. 12 panel A illustrates device 1210 used for aliquoting
suspension with five fluidic channels (1211, 1212, 1213, 1214, and
1215) joined at junction 1240. Fluidic channels 1211, 1212, and
1213 carried fluid toward junction 1240, whereas fluidic channels
1214 and 1215 carried fluid away from junction 1240. Solenoid valve
1220 was connected to port 1216 in fluidic communication with
channel 1211 via tubing 1221, and solenoid valve 1222 was connected
to port 1217 in fluidic communication with channel 1212 via tubing
1223.
[0278] Solenoid valves 1220 and 1222 was actuated to close or open
with electronic or computer signal (e.g. TTL signal). When solenoid
valve 1220 was closed while solenoid valve 1222 remained open,
aliquot 1260 of blood containing rare cells in the incoming channel
1213 was diverted to the left outlet channel 1214 (see panel B, 0
ms). When solenoid valve 1220 was opened while solenoid valve 1222
remained closed, aliquot 1260 of blood containing rare cells in the
incoming channel 1213 was diverted to the right outlet channel 1215
(see panel C, 2 ms). Aliquot redirecting using a combination of
solenoid valves 1220 and 1222 could channel the aliquot from one
channel to another in as little as 2 ms.
[0279] L. Example 12
[0280] To test the performance of eDAR device, a mixture of blood
and cancer cells was prepared according to the following procedure:
.times.10E6/mL MCF-7 cells were labeled with 20 .mu.L, of
fluorescent EpCAM antibody. This cell mixture was then diluted to
1.times.10E5 cells/mL with Isoton hematological diluent. Ten .mu.L,
of the diluted cell mixture was then added to 2 mL of whole human
blood and flowed through the aliquoting device. The flow rate in
the aliquoting device was nominally 30 .mu.L/min unless otherwise
indicated.
[0281] FIG. 13 shows the fluorescence signals collected from 2
avalanche photodiodes (APDs) positioned at different locations
upstream and downstream of junction 1116 (or 1240). Plot 1310 shows
the signal trace 1311 from one APD configured to detect the
presence of EpCAM molecule in an aliquot at detection volume 1140
(or 1270), whereas Plot 1320 shows the signal trace 1321 from a
second APD configured to detect the presence of EpCAM molecule in
channel 1114 (or 1214). The signal peaks 1312 matched substantially
the signal peaks 1322, indicating that channeling of aliquot was
correct, resulting in a high recovery of rare cells.
[0282] To further investigate the performance of eDAR device, the
number of cancer cells directed into the correct flow channel were
counted and subsequently collected ("recovered") while adjusting
the length of time the solenoid valve (1220, 1222) or piston (1120,
1121) remain closed or open ("pulse length"). The percentage
recovery was computed by dividing the number of rare cells
collected in the correct channel by the number of rare cells
detected by an APD at detection volume 1140 or 1270. FIG. 14 Plot
1410 shows the percentage of cancer cells recovered as a function
of the pulse length. Trace 1420 indicates that when the pulse width
was 10 ms or below, 100% of the cancer cells were collected in the
correct channel. As pulse width increased, trace 1420 decreased,
indicating a loss of cells to the wrong channel.
[0283] By adjusting the flow rate of incoming channel 1113 (or
1213), we also observed that the recovery could be optimized. FIG.
15 shows plot 1510 with trace 1520 indicating the percentage of
rare cells recovered as a function of incoming flow rate. When the
flow rate was below 30 .mu.L/min, the recovery was between 89-100%.
However, as the flow rate increased, the recovery decreased,
indicating an increasing loss of rare cells in the wrong
channel.
[0284] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
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