U.S. patent application number 15/934018 was filed with the patent office on 2018-08-02 for analysis, secure access to, and transmission of array images.
This patent application is currently assigned to BioArray Solutions, Ltd.. The applicant listed for this patent is BioArray Solutions, Ltd.. Invention is credited to Chiu CHAU, Scott DETERMAN, Michael SEUL, Xiongwu XIA.
Application Number | 20180218498 15/934018 |
Document ID | / |
Family ID | 32326436 |
Filed Date | 2018-08-02 |
United States Patent
Application |
20180218498 |
Kind Code |
A1 |
SEUL; Michael ; et
al. |
August 2, 2018 |
ANALYSIS, SECURE ACCESS TO, AND TRANSMISSION OF ARRAY IMAGES
Abstract
Systems and methods are provided the autocentering,
autofocusing, acquiring, decoding, aligning, analyzing and
exchanging among various parties, images, where the images are of
arrays of signals associated with ligand-receptor interactions, and
more particularly, ligand-receptor interactions where a multitude
of receptors are associated with microparticles or microbeads. The
beads are encoded to indicate the identity of the receptor
attached, and therefore, an assay image and decoding image are
aligned to effect the decoding. The images or data extracted from
such images can be exchanged between de-centralized assay locations
and a centralized location where the data are analyzed to indicate
assay results. Access to data can be restricted to authorized
parties in possession of certain encoding information, so as to
preserve confidentiality.
Inventors: |
SEUL; Michael; (Fanwood,
NJ) ; XIA; Xiongwu; (Dayton, NJ) ; CHAU;
Chiu; (Edison, NJ) ; DETERMAN; Scott; (North
Andover, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioArray Solutions, Ltd. |
Warren |
NJ |
US |
|
|
Assignee: |
BioArray Solutions, Ltd.
Santa Clara
CA
|
Family ID: |
32326436 |
Appl. No.: |
15/934018 |
Filed: |
March 23, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15010597 |
Jan 29, 2016 |
9928587 |
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15934018 |
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14202357 |
Mar 10, 2014 |
9251583 |
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15010597 |
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13084869 |
Apr 12, 2011 |
8712123 |
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14202357 |
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11439599 |
May 23, 2006 |
7940968 |
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13084869 |
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10714203 |
Nov 14, 2003 |
7526114 |
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11439599 |
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60426839 |
Nov 15, 2002 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G06T 7/11 20170101; G06K
9/00 20130101; G01N 33/566 20130101; G06K 2209/07 20130101; G06T
2207/30072 20130101; G06K 9/00147 20130101; G06T 2207/20152
20130101; G06T 7/33 20170101; G06T 7/0012 20130101; G06T 7/155
20170101; G06T 2207/10064 20130101 |
International
Class: |
G06T 7/00 20060101
G06T007/00; G06T 7/155 20060101 G06T007/155; G01N 33/566 20060101
G01N033/566; G06K 9/00 20060101 G06K009/00; G06T 7/11 20060101
G06T007/11; G06T 7/33 20060101 G06T007/33 |
Claims
1. (canceled)
2. A method of determining alignment between two or more images,
where each image is of a correspondingly arranged array having
corresponding subsets of distinguishable signals, comprising:
establishing a two-dimensional grid having units, each unit
corresponding to a particular position in the arrays from which a
signal originates, and aligning the two grids such that
corresponding signals align in a one-to-one correspondence such
that a detectable difference results when one array is shifted out
of alignment by one or more grid units with respect to the other
array.
3. The method of claim 2, wherein the detectable difference results
from variation in the correspondence of one or more of the
subsets.
4. The method of claim 3, whrein the signals are optical signals
and the additional signals can be seen by viewing the arrays in
another color channel.
5. The method of claim 2, wherein different signals in the array
originate from beads, and different beads occupy different
positions in the array.
6. The method of claim 5, wherein the arry of beads includes
differently colored beads, and the colors are detected.
7. The method of claim 2 performed by carrying out the steps of the
following pseudocode: TABLE-US-00010
LoadDecodingData(DecodingDataRecord, Decoding Map);
LoadAssayData(AssayDataRecord,Intensity Array); MinVariance =
-1000; MinVarianceLocation = 0; /**Check Variance Produced by 7
Pssible Unit Displacements - see Text**/ For (i=0; i<7; i++){
SiftGridPosition(i); FilterDarkBeads( ); Variance[i] =
Merge(DecodingMap, Intensity Array); IF(Variance[i] <
MinVariance){ MinVariance = Variance[i]; MinVarianceLocation = i; }
}; WriteAssayData[(MinVarianceLocation, AssayDataRecord); /**Save
Location**/
8. The method of claim 7, wherein the pseudocode is performed by a
programmed computer.
9. A method of image segmentation producing a partition of an image
of a planar array of objects, comprising: determining overall
orientation of the array using a set of reference lines which are
brought into alignment with symmetry axes of the array; replicating
the reference lines by shifting normal to each symmetry direction
and finding the shortest path connecting the starting point and
point of each shifted reference line; replicating the replicating
step until the replicated reference line falls outside the array
boundary.
10. The method of claim 9, wherein the overall orientation is
determined using horizontal and vertical reference lines of known
length and computing the shortest path from the starting and end
point of each reference line, the path following the local
intersection minimal path.
11. The method of claim 10, whrein the minimal path is the path of
shortest length following the locus of local intensity minima.
12. The method of claim 9, wherein the partition is in the form of
a mesh, each mesh field delineating one object.
13. The method of claim 9, wherein objects are color-encoded beads
fiunctionalized with receptors capable of binding ligands contained
in an analyte solution.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/084,869, filed Apr. 12, 2011, which is a
continuation of U.S. patent application Ser. No. 11/439,599, filed
May 23, 2006, now issued as U.S. Pat. No. 7,940,968, which is a
continuation of U.S. patent application Ser. No. 10/714,203, filed
Nov. 14, 2003, now issued as U.S. Pat. No. 7,526,114, which claims
priority to U.S. Provisional Application No. 60/426,839, filed Nov.
15, 2002. All of the applications and patents cited above are
hereby incorporated by reference in their entireties.
BACKGROUND
[0002] Recent rapid advances in molecular biology have created more
demand for high volume testing based on the need to screen ever
larger compound libraries, validate ever increasing numbers of
genetic markers and test ever more diversified patient populations.
This has led to the development of new array formats, particularly
for nucleic acid and protein-protein interaction analysis, which
increase parallel processing by performing requisite assays in a
"multiplexed" format.
[0003] Conventionally, such assays are performed by producing
arrays of nucleic acids and antibodies by way of "spotting" or
"printing" of aliquot solutions on filter paper, blotting paper or
other substrates. However, notwithstanding their widespread current
use in academic research targeting gene expression and protein
profiling, arrays produced by spotting have shortcomings,
particularly in applications placing high demands on accuracy and
reliability and where large sample volume and high throughput is
required. In another more recently developed technique, spatially
encoded probe arrays are produced by way of in-situ photochemical
oligonucleotide synthesis. However, this technology is limited in
practice to producing short oligonucleotide probes--and requiring
alternative technologies for the production of cDNA and protein
arrays--and precludes rapid probe array customization given the
time and cost involved in the requisite redesign of the
photochemical synthesis process.
[0004] In addition to these inherent difficulties in assay
performance, spatially encoded arrays produced by methods of the
art generally produce data of such poor quality that specialized
scanners are required to extract data of useable quality.
Commercial systems available for this purpose require confocal
laser scanning--a slow process which must be repeated for each
desired signal color--and limit the spatial resolution to .about.5
.mu.m.
[0005] In order to resolve many of the problems associated with
diagnostic and analytical uses of "spotted arrays" of
oligonucleotides and proteins (as outlined in "Multianalyte
Molecular Analysis Using Application-Specific Random Particle
Arrays," U.S. application Ser. No. 10/204,799, filed on Aug.
23/2002; WO 01/98765), arrays of oligonucleotides or proteins
arrays can be formed by displaying these capture moieties on
chemically encoded microparticles ("beads") which are then
assembled into planar arrays composed of such encoded
functionalized carriers. See U.S. patent application Ser. No.
10/271,602 "Multiplexed Analysis of Polymorphic Loci by Concurrent
Interrogation and Enzyme-Mediated Detection," filed Oct. 15, 2002,
and Ser. No. 10/204,799 supra.
[0006] Microparticle arrays displaying oligonucleotides or proteins
of interest can be assembled by light-controlled electrokinetic
assembly near semiconductor surfaces (see, e.g., U.S. Pat. Nos.
6,468,811; 6,514,771; 6,251,691) or by a direct disposition
assembly method (previously described in Provisional Application
Ser. No. 60/343,621, filed Dec. 28, 2001 and in U.S. application
Ser. No. 10/192,352, filed Jul. 9, 2002).
[0007] To perform nucleic acid or protein analysis, such encoded
carrier arrays are placed in contact with samples anticipated to
contain target polynucleotides or protein ligands of interest.
Capture of target or ligand to particular capture agents displayed
on carriers of corresponding type as identified by a color code
produces, either directly or indirectly by way of subsequent
decoration, in accordance with one of several known methods, an
optical signature such as a fluorescence signal. The identity of
capture agents including probes or protein receptors (referred to
herein sometimes also collectively as "receptors") generating a
positive assay signal can be determined by decoding carriers within
the array.
[0008] These microparticle arrays can exhibit a number of
spectrally distinguishable types of beads within an area small
enough to be viewed in a microscope field, It is possible to
achieve a high rate of image acquisition because the arrays obviate
the need for confocal laser scanning (as used with spotted or
in-situ synthesized arrays) and instead permit the use of direct
("snapshot") multicolor imaging of the entire array under a
microscope. If the system could be automated further, such that,
for example, the microscope is automatically repositioned to
optimally capture images from multiple arrays present on a
multichip carrier and to positions optimizing decoding of the
array, this would facilitate unattended acquisition of large data
lots from multiplexed assays.
[0009] In one format using microbead arrays, the encoding capacity
of a chip (which includes several distinct subarrays) can be
increased even where using the same set of color codes for the
beads in each subarray. When subarrays are spatially distinct, the
encoding capacity becomes the product of the number of bead colors
and the number of subarrays.
[0010] In order to match the rates of data acquisition enabled by
direct imaging, rapid and robust methods of image processing and
analysis are required to extract quantitative data and to produce
encrypted and compact representations suitable for rapid
transmission, particularly where there is off-site analysis and
data storage. Transmission of data should be secure, and should be
accessible only by authorized parties, including the patient but,
because of privacy concerns, not to others.
SUMMARY
[0011] Disclosed are methods of increasing the confidence of the
analysis, and for rapid and automated decoding of encoded arrays
used in assays, assay data recorded in the form of images generated
from arrays of ligand-receptor interactions; and more particularly,
where different receptors are associated with different encoded
microparticles ("beads"), and results are determined upon decoding
of the arrays. Also disclosed are methods for transmitting and
archiving data from such assay arrays in a manner such that access
is limited to authorized persons, and such that the chance of
assigning one patient's results to another are minimized. These
methods are particularly useful where assays are performed at
decentralized user ("client") sites, because the methods permit
secure exchange of data between the client and a central facility
("information keeper"), where the data can be centrally decoded and
analyzed so as to provide greater reliability, and then archived in
a restricted manner where only authorized users have access.
[0012] In a centralized regime, patient samples, collected in the
field, are sent for analysis to a central location, where they are
assayed. Results are provided to authorized users by remote
transmission. Users, while relieved of any responsibility relating
to assay completion and data analysis, are faced with the loss of
control over the assay implementation and analysis and may face the
inconvenience of significant delay. Non-standard assays may be
unavailable or prohibitively expensive. In addition, this service
ordinarily will not be suitable for perishable samples, or large to
collections of samples, such as those created in a pharmaceutical
research laboratory.
[0013] In a decentralized paradigm, analytical instrumentation,
such as microplate readers complete with all requisite software,
are distributed to users who perform assays, record results, and
may also perform subsequent data analysis. Alternatively, assay
results may be transmitted to a central facility for decoding,
analysis, processing and archiving, and such centralized procedures
may provide greater reliability.
[0014] The analysis server model (useful, inter alia, for molecular
diagnostics), as disclosed herein, expands upon these paradigms by
combining decentralization of assay performance with centralized
data analysis. That is, while assay performance and data generation
are at user facilities, critical aspects of subsequent data
analysis and related services may be performed in a centralized
location which is accessible to authorized users in a two-way mode
of communication via public or private computer networks. The
analysis server model can be applied to assays performed in a
highly parallel array format requiring only a simple imaging
instrument, such as a microscope, to record complex assay data, but
requiring advanced methods of analysis and mathematical modeling to
reliably process and analyze assay data. Images, recorded at a user
location, are uploaded to a centralized location where such
analysis are performed, results being made available to authorized
users in real time.
[0015] The methods and processes are further explained below with
reference to the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 displays a 1.times.8 multichip carrier, where chips
with encoded arrays on a surface are housed in the carrier's
wells.
[0017] FIG. 2 is a flowchart of an imaging system, including image
acquisition, a decoder and a reader for image processing, and an
analyzer for data analysis.
[0018] FIG. 3 is a flowchart illustrating an exemplary system where
assay data can be decoded and centrally analyzed.
[0019] FIG. 4 is a flowchart illustrating viewing a multiplexed
assay and recording of the assay image.
[0020] FIG. 5 is a flowchart depicting sample collection,
performing of assays and acquisition of an assay image.
[0021] FIG. 6 is a flowchart of the formation of a decoding image
and decoding data records where the resulting information can be
stored in a database.
[0022] FIG. 7 is a diagram illustrating the, reading and processing
of an assay image and recording of an assay data record, where the
resulting information can be stored in a database.
[0023] FIG. 8 is a flowchart illustrating the recording, analysis
and decoding of assay data, and combining with an assay data record
to generate a decoded assay data record, where the resulting
information can be stored in a database.
[0024] FIG. 9A shows a grid (with extra rows and columns) in
unshifted position (0,0).
[0025] FIG. 9B shows the grid of FIG. 9A and in a configuration
shifted to position (1,1).
[0026] FIG. 9C illustrates the seven possible shifting positions
for the grid of 9A and 9B.
[0027] FIG. 10A is an illustration of a two-party, multiple-user,
data exchange.
[0028] FIG. 10B illustrates a three-party data exchange, where one
party is the patient.
[0029] FIG. 10C illustrates more specifically a secure data
transaction between three parties, where one party is the
patient,
[0030] FIGS. 11A to 11D depict the DECODER Graphical. User
Interface (GUI) illustrating steps in the course of processing a
decoding image.
[0031] FIG. 12 illustrates a constructed Decoding Map.
[0032] FIGS. 13A and 13B show a READER Graphical User Interface
(GUI) illustrating steps in the course of processing a assay
image.
[0033] FIG. 14 illustrates a decoded assay data record in histogram
form.
[0034] FIG. 15A illustrates mesh overall orientation with the
shortest path algorithm, wherein L.sub.h.sup.ref and
L.sub.v.sup.ref are the reference lines. By applying shortest path,
L.sub.h and L.sub.v are found. It can be seen that when L.sub.h is
shorter than L.sub.v, then the overall orientation of the mesh is 0
degrees.
[0035] FIG. 15B illustrates vertical line grid partition by the
shortest path algorithm.
DETAILED DESCRIPTION
[0036] FIG. 1 depicts a multichip carrier (100) displaying a
barcode (110) along with a set of 2.times.2 chips (120) in each of
eight positions, each such chip comprising one or more random
array(s) of encoded microparticles (130). The decoding of the
microparticles 130 and analysis and transmission of the data can be
performed in accordance with the methods described herein. Each of
the 2.times.2 chip subarrays (i.e., four chips) correspond with one
individual patient sample. Thus, given a set of 128 distinguishable
colors for the beads in the array, this can produce an array
complexity of 4.times.128=512 for each individual. The 1.times.8
sample format shown in FIG. 1 with a center-to-center distance of 9
mm matches the standard 8.times.12 microwell format and is readily
scaled to n.times.8 for high-throughput sample handling (as
described further in U.S. patent application Ser. No.
10/192,352).
[0037] As illustrated in FIG. 2, the imaging system comprises
hardware for acquiring a decoding image (200), means for acquiring
an assay image (210), a decoder (220), a reader (230), and an
analyzer (240). Acquiring and recording a decoding image or an
assay image can be accomplished using the system shown in FIG.
4.
[0038] Molecular interaction analysis on a random array of encoded
microparticles or beads, where each subpopulation of such encoded
beads displays a unique receptor molecule, for example, an
oligonucleotide, or protein molecule, can be performed using these
systems. Each such receptor is designed so as to be able to form a
molecular complex with a cognate target analyte or ligand, the
formation of a complex resulting in an, optical signature (for
example, the ligand can be fluorescently labeled and thus
detectable following complex formation) that is detected by
acquiring and analyzing images of the array. Assay results take the
form of an image comprising a set of intensities, each of which is
uniquely associated with one receptor-ligand interaction in the
array. Such assays are well-suited for DNA and protein analysis
performed in a multiplexed or parallel mode using an array format,
including applications such as genetic expression profiling,
polymorphism and mutation analysis, protein-protein analysis
including antibody-antigen interaction analysis, organic
compound-receptor interactions, and further including all those
disclosed in PCT/US01/20179, U.S. Pat. Nos. 6,251,691; 6,387,707
and U.S. patent application Ser. No. 10/271,602, all of which are
incorporated herein by reference.
[0039] The decoding of encoded arrays and the analysis,
interpretation or storage of assay results requiring access to
methods and algorithms or access to databases which are not
available at the location housing the analytical instrumentation,
but are instead available at a remote location, is addressed
herein. Special consideration is given to situations in which these
operations, or parts thereof such as the collection and preparation
of a sample, the actual assay or assays of interest and additional
operations such as interpretation or consultation are performed in
separate locations. In one embodiment, the methods and apparatus
described herein permit complex multianalyte analysis to be
performed in locations visited by a patient whose sample is
collected and analyzed on site, while providing transaction
protocols to perform data analysis and optional additional
services, such as data interpretation and archiving to be performed
off-site. The methods herein permit the secure exchange of
information recorded on site and analyzed at the site of an
application service provider.
[0040] Accordingly, the systems and methods provided herein for the
analysis of clinical samples or other analytes provide for
communication among three (or optionally more) participants,
including: (i) the sample originator, for example a patient seeking
clinical diagnosis or a biomedical research laboratory seeking
analytical services; (ii) the analysis provider (which may or may
not also be the tester performing the assay); and (iii) the tester,
which performs the assay but is generally provided minimal
information about assay outcomes, decoding, or sample origination;
and (iv) an optional intermediary, for example a sample collector
and/or processor or communicator of results, such as a
counselor.
[0041] These methods allow the rapid assaying and analysis of
customized random encoded bead arrays, where a multiplexed assays
are performed on patient samples, and multiple assays may be
conducted. Suitable panels may include, for example, a tumor marker
panel including antigens such as PSA and other suitable tumor
markers, an allergy panel, a pregnancy panel comprising tests for
human chorionic gonadotropin, hepatitis B surface antigen, rubella
virus, alpha fetoprotein, 3' estradiol and other substances of
interest for monitoring a pregnant individual; a hormone panel, an
autoimmune disease panel including tests for rheumatoid factors and
panel reactive antibodies and other markers associated with
autoimmune disorders, a panel of blood-borne viruses and a
therapeutic drug panel comprising tests for Cyclosporin, Digoxin
and other therapeutic drugs of interest.
[0042] In addition, such panels also may include, for example,
oligonucleotide probes designed for nucleic acid analysis including
analysis of cDNA panels for gene expression profiling,
oligonucleotide probe panels designed for the multiplexed analysis
of mutations causing genetic diseases such as cystic fibrosis,
Tay-Sachs disease, Ashkenazi Jewish diseases including Gaucher
disease and others, the analysis of polymorphisms such as those in
the Human Leukocyte Antigen complex which determine the degree of
compatibility between donor and recipient in transplantation of
bone marrow or solid organs, a blood antigen panel for blood
typing, the analysis of chromosomal aberrations such as those
underlying Down Syndrome and others surveyed in prenatal screening
or certain blood-borne cancers such as certain leukemias. The
multiplexed nucleic acid analysis involved in assaying of these
panels can be performed using either hybridization-mediated
detection or hybridization-mediated elongation-mediated detection,
as described in U.S. patent application Ser. No. 10/271,602,
entitled: "Multiplexed Analysis of Polymorphic Loci by Concurrent
Interrogation and Enzyme-Mediated Detection" filed Oct. 15,
2002.
[0043] In either hybridization-mediated detection or
hybridization-mediated elongation-mediated detection, an
association of polynucleotide in the sample with a probe
oligonucleotide on a bead results in an assay signal in the form of
an optical signature. For example, in the READ.TM. format, each
encoded bead within the array (where each bead has multiple probes
attached thereto) may produce one or more of such optical
signatures which are able to be recorded by the systems of the
invention. The optical signature can be a fluorescence signature.
Optical signatures of interest include, without limitation,
luminescence including bioluminescence, chemiluminescencve and
electrochemiluminescence. Direct visual signatures resulting, for
example, from the transformation of the assay locus, for example,
by agglutination of multiple beads, or the attachment of marker
particles to assay loci, can also be recorded and analyzed using
the methods set forth herein.
I. Automated High-Throughput Array Imaging for Molecular
Interaction Analysis
I.1 Assay and Decoding Data Records
[0044] FIG. 3 is an illustration of the methods to conduct the
analysis of analytes using bead arrays assembled on substrates,
according to the READ process of multiplexed analysis. The method
of FIG. 3 involves producing a bead array (300), obtaining a
decoding image (310), processing the decoding image using a decoder
(320), and obtaining a decoding data record (330). In parallel, an
assay is performed using the bead array (340) to obtain an assay
image (350), where the assay image is processed using a reader
(321) and the assay data is recorded (360). The decoding data and
assay data are then combined and the image is analyzed (370). The
decoding image may represent a combination of several distinct
images recorded in separate spectral bands or color channels, where
the encoding is with chemically and/or physically distinguishable
characteristics that uniquely identify the binding agent displayed
on the bead surface. For example, when the uniquely distinguishable
characteristic is color, a "decoding image" of the bead array
(where the bead are immobilized on the substrate) is recorded to
reference the color code of constituent beads, where the color code
uniquely corresponds to the chemical identity of the binding agents
displayed on each individual bead's surface.
[0045] Referring to FIG. 3, the decoding image (310) may be
generated following completion of array assembly by the array
manufacturer at the manufacturing site or it may be generated by
the user of the array in connection with the completion of a
bioassay or other chemical test, either prior to, or subsequent to
completion of the assay or test at the test site. The decoding
image (310) becomes part of the decoding data record (330), which
also contains a variety of identifiers for reagent, microparticle
and substrate batch and lot numbers. The decoding image (310) can
be generated following array assembly at the manufacturing site and
processed to create a decoding data record (330) which is stored in
a database on a central server. Access to the decoding data record,
or parts thereof, can be accessible in the form of a copy the
record, for example, of copies on a recording medium such as a CD
that is distributed along with arrays and multichip carriers or
cartridges. Alternatively, access to the decoding data record, or
parts thereof; is made accessible in the form of authenticated
database accessible only to authorized users as disclosed
herein.
[0046] Following completion of an assay (340) at a user site, the
assay image is recorded (350) and an Assay Data Record (360) is
created which serves to record the optical signature(s) indicating
the binding of ligand molecules to immobilized receptors. For
example, when fluorescence is selected to provide the optical
signature of interest, the fluorescence intensity recorded from
each position within the array indicates the amount of complex
formed in that location by receptor and ligand binding or
hybridization. Multiple modes of generating such optical signatures
include the direct or indirect labeling of target analytes (for
example, by using fluorescent primers to conduct PCR of genomic
regions to be assayed) or the introduction of fluorescence by way
of probe elongation using labeled nucleotides. See, e.g., U.S.
patent application Ser. No. 10/271,602 "Multiplexed Analysis of
Polymorphic Loci by Concurrent Interrogation and Enzyme-Mediated
Detection." The assay image forms the assay data record.
[0047] As described herein below, the methods herein can be used
for processing decoding and assay images, for extracting
representations suitable for rapid transmission and for rapidly and
reliably combining decoding and assay image signatures so as to
associate assay results recorded from specific array locations with
corresponding chemically encoded probe identities.
[0048] Assembly of Random Encoded Bead Arrays. Random encoded
arrays may be assembled by the methods described in U.S. Pat. No.
6,251,691, or in U.S. patent application Ser. No. 10/192,352,
entitled "Arrays of Microparticles and Methods of Preparation
Thereof," incorporated herein by reference in its entirety. These
methods combine separate batch processes that respectively serve to
produce application-specific substrates (e.g., chips at the wafer
scale) and encoded bead libraries whose constituent beads are
functionalized (e.g., at the scale of .about.10.sup.8 beads/100 ul
of suspension) to display receptors, such as nucleic acids and
proteins of interest. Beads assembled in an array may be
immobilized by physical or chemical means to produce fixed random
encoded arrays.
[0049] In addition, the methods described in U.S. Pat. No.
6,251,691 may be used to form multiple bead arrays. Alternatively,
multiple bead arrays can be formed simultaneously in discrete fluid
compartments maintained on the same chip. The integration of array
assembly with microfluidics produces a self-contained,
miniaturized, optically programmable platform for parallel protein
and DNA analysis. Once formed, these multiple bead arrays may be
used for concurrent processing of multiple samples.
[0050] Spatial encoding of multiple arrays also can be accomplished
by assembling planar bead arrays in a desired location, using
discrete fluid compartments or the assembly methods described in
U.S. Pat. No. 6,251,691 Alternatively, spatial encoding can be
accomplished by assembling separate chips, each carrying at least
one random encoded array drawn from a specific pool, into a
designated configuration of multiple chips.
[0051] Chemical Encoding and Functionalization of Beads. Chemical
encoding may be accomplished by staining beads with sets of
optically distinguishable tags, such as those containing one or
more fluorophore dyes spectrally distinguishable by excitation
wavelength, emission wavelength, excited-state lifetime or emission
intensity. Two-color and three-color combinations, where the latter
may be constructed as "stacked" two-color combinations, are decoded
as described herein.
[0052] The optically distinguishable tags may be used to stain
beads in specified ratios, as disclosed, for example, in Fulwyler,
U.S. Pat. No. 4,717,655, which is incorporated herein by reference
in its entirety. Staining may also be accomplished by swelling of
particles in accordance with methods known to those skilled in the
art (see, e.g., Molday, Dreyer, Rembaum & Yen, J. Mol Biol 64,
75-88 (1975); L. Bangs, "Uniform latex Particles, Seragen
Diagnostics, 1984). Beads can be encoded by swelling and bulk
staining with two or more colors, each individually at separate
intensity levels, and mixed in separate nominal molar ratios in
accordance with methods known to the art. See also U.S. patent
application Ser. No. 10/348,165, entitled: Method of Controlling
Solute Loading of Polymer Microparticles; filed Jan. 21, 2003.
Combinatorial color codes for exterior and interior surfaces is
disclosed PCT/US98/10719, which is incorporated herein by
reference.
[0053] Beads to be used in the bead arrays of the invention for
biomolecular analysis are functionalized by a binding agent
molecule attached thereto, where the molecule may be, for example,
DNA (oligonucleotides) or RNA, fragments, peptides or proteins,
aptamers and small organic molecules attached in accordance with
processes known in the art, e.g., with one of several coupling
reactions of the known art (G. T. Hermanson, Bioconjugate
Techniques (Academic Press, 1996); L. Ilium, P. D. E. Jones,
Methods in Enzymology 112, 67-84 (1985)). The binding agent
molecule may be covalently attached to the bead. Beads may be
stored in a buffered bulk suspension until needed.
[0054] Functionalization typically may be performed, for example,
with a one-step or two-step reaction which may be performed in
parallel using standard liquid handling robotics and a 96-well
format to covalently attach any of a number of desirable
functionalities to designated beads. Beads of core-shell
architecture may be used (as described in U.S. Provisional
Application entitled: "Ionic Gel-Shell Beads with Adsorbed or Bound
Biomolecules," filed Oct. 28, 2003, and applications claiming
priority thereto) where the shell is a polymeric layer. Samples may
be drawn along the way for automated QC measurements. Each batch of
beads preferably has enough members such that chip-to-chip
variations with different beads on chips are minimized.
[0055] Beads may be subjected to quality control (QC) steps prior
to array assembly, for example, the determination of morphological
and electrical characteristics, the latter including surface
("zeta") potential and surface conductivity. In addition, assays
may be performed on beads in suspension before they are introduced
to the substrate, to optimize assay conditions, for example, to
maximize assay sensitivity and specificity and to minimize
bead-to-bead variations. QC steps for substrates may include
optical inspection, ellipsometry and electrical transport
measurements.
[0056] Substrates. Substrates, e.g., silicon wafers and chips, are
used which may be patterned by invoking standard methods of
semiconductor processing, for example to it) implement interfacial
patterning methods of LEAPS by, e.g., patterned growth of oxide or
other dielectric materials to create a desired configuration of
impedance gradients in the presence of an applied AC electric
field. See U.S. Pat. No. 6,251,691. Patterns may be designed so as
to produce a desired configuration of AC field-induced fluid flow
and corresponding particle transport, or to trap particles in
wells, as described in US Provisional Application entitled:
Immobilization of Bead-displayed Ligands on Substrate Surfaces,"
filed Jun. 12, 2003, Ser. No. 60/478,011.
[0057] In addition, substrates may be compartmentalized by
depositing a thin film of a UV-patternable, optically transparent
polymer to affix to the substrate a desired layout of fluidic
conduits and compartments to confine fluid in one or several
discrete compartments, thereby accommodating multiple samples on a
given substrate. Other substrates such as patternable or machinable
ceramics also are suitable.
[0058] Customization by Pooling. Bead-displayed probes of interest
can be selected from a library of beads and pooled prior to array
assembly. That is, customization of assay composition is achieved
by selecting aliquots of designated encoded beads from individual
reservoirs in accordance with a specified array composition.
Aliquots of pooled suspension are dispensed onto a selected
substrate (e.g., a chip). The aliquots may be mixed or may be
separated to form a multiplicity of planar random subarrays of
encoded beads, each subarray representing beads drawn from a
distinct pool. The array may be laid out in a manner such that
aliquot positions in the array correspond to the identity of each
aliquot of the pooled bead population.
[0059] Array Analysis. The binding interaction between the receptor
(which may be an oligonucleotide) displayed on color-encoded
functionalized beads and a ligand (or "analyte") may be analyzed
after a random encoded bead array is assembled in a designated
location on the substrate or chip. For example, bead arrays may be
formed after completion of the assay, subsequent to which an assay
image and a decoding image may be taken of the array.
[0060] Immobilization Microparticle arrays may be immobilized by
mechanical, physical or chemical anchoring as described in
PCT/US01/20179 (counterpart of U.S. patent application Ser. No.
10/192,352), including by trapping particles in wells, as described
in US Provisional Application entitled: Immobilization of
Bead-displayed Ligands on Substrate Surfaces," filed Jun. 12/2003,
Ser. No. 60/478,011.
[0061] In certain embodiments, bead arrays may be immobilized by
physical adsorption mediated by application of a DC voltage, set to
typically <5V (for beads in the range of 2-6 Ym, a gap size of
100-150 Tm, and a silicon oxide layer of .about.100 Angstrom
thickness). Application of such a DC voltage for <30 s in
"reverse bias" configuration--so that an n-doped silicon substrate
would form the anode--causes bead arrays to be permanently
immobilized. See U.S. Pat. No. 6,251,691.
[0062] In certain embodiments, the particle arrays may be
immobilized by chemical means, e.g., by forming a composite
gel-particle film. In one exemplary method for forming such
gel-composite particle films, a suspension of microparticles is
provided which also contain all ingredients for subsequent in-situ
gel formation, namely monomer, crosslinker and initiator. The
particles may be assembled into a planar assembly on a substrate by
application of the LEAPS.TM. process, as described in U.S. Pat. No.
6,251,691. Following array assembly, and in the presence of the
applied AC voltage, polymerization of the fluid phase is triggered
by thermally heating the cell .about.40-45.degree. C. using an IR
lamp or photometrically using a mercury lamp source, to effectively
entrap the particle array within a gel. Gels may be composed of a
mixture of acrylamide and bisacrylamide of varying monomer
concentrations, from about 20% to 5% (acrylamide:
bisacrylamide=37.5: 1, molar ratio), or, in the alternative, any
other low viscosity water soluble monomer or monomer mixture may be
used as well. In one example, thermal hydrogels are formed using
azodiisobutyramidine dihydrochloride as a thermal initiator at a
low concentration ensuring that the overall ionic strength of the
polymerization mixture falls in the range of .about.0.1 mM to 1.0
mM. The initiator used for the UV polymerization is Irgacure
2959.RTM. (2-Hydroxy-4'-hydroxyethoxy-2-methylpropiophenone, Ciba
Geigy, Tarrytown, N.Y.). The initiator is added to the monomer to
give a 1.5% by weight solution. The methods described in U.S.
patent application Ser. No. 10/034,727 are incorporated herein by
reference.
[0063] In certain embodiments, the particle arrays may be
immobilized by mechanical means, for example, such arrays may be
placed into an array of recesses may be produced by standard
semiconductor processing methods in the low impedance regions of
the silicon substrate. The particle arrays may moved into the
recesses by, e.g., utilizing LEAPS.TM.-mediated hydrodynamic and
ponderomotive forces to transport and accumulate particles in
proximity to the recesses. The A.C. field is then switched off and
particles are trapped into the recesses and mechanically confined.
Excess beads are removed leaving behind a geometrically ordered
random bead array on the substrate surface.
[0064] Carriers and Cartridges. Substrates (e.g., chips) with
immobilized bead arrays may be placed in distinct enclosed
compartments, and samples and reagents may be transported in and
out of the compartments by means of fluidic interconnection.
On-chip immunoassays, including those for various cytokines, e.g.,
interleukin (IL-6) may be performed in this format. In such
immunoassays, samples are allowed to react with beads immobilized
on the chip and adsorption of targets in the samples by the
receptors on the beads may be detected by binding of fluorescently
labeled secondary antibodies.
[0065] Random Encoded Array Detection. Once the functionalized and
encoded beads are prepared, and assembled on the substrate, a
binding assay may be performed. The array can function as a
two-dimensional affinity matrix which displays receptors or binding
agents (e.g., oligonucleotides, cDNA, aptamers, antibodies or other
proteins) to capture analytes or ligands (oligonucleotides,
antibodies, proteins or other cognate ligands) from a solution or
suspension that is brought in contact with the array. The bead
array platform may be used to perform multiplexed molecular
analysis, such as, e.g., genotyping, gene expression profiling,
profiling of circulation protein levels and multiplexed kinetic
studies, and may be used for the implementation of random encoded
array detection (READ.TM.), including analysis based on image
acquisition, processing and analysis.
I.2 Multicolor Image Acquisition Using an Automated Array Imaging
System
[0066] Multicolor images can be used to encode and display
information recorded in two or more color channels. The
construction of multicolor images can be accomplished by merging
two or more images recorded in separate spectral bands and
distinguished by selection of suitable color filter combinations,
as is well-known in the art.
[0067] The READ.TM. format provides for multicolor images before
and after the assay, referred to respectively as a decoding image
and an assay image. The decoding image serves to record the
location of particular identified solid phase carriers--and hence
the identity of receptors displayed on such carriers--based on
their color-encoding in an array. These solid phase carriers may be
color encoded using, for example, combinations of two or more
fluorescent dyes. The assay image reflects the optical signatures
induced by association of target analytes with carrier-displayed
receptors. In one example, useful in gene expression profiling,
signal produced on an array by the hybridization of cDNA or RNA
produced from a tissue sample of interest and labeled with
fluorescent dye (e.g., Cy5) may be compared with the signal
produced from a known quantity or concentration of cDNA or RNA
produced from a reference sample and labeled with a fluorescent
dye, (e.g., Cy3 or Cy5). The comparison of signal from the tissue
sample and the reference sample indicates the level of gene
expression. An analogous format may be used in molecular
cytogenetics applications. Protein-protein interactions can also be
monitored with this format, where the protein in the sample is
labeled following its association with the bead-bound protein in
the array, for example, by using a labeled antibody which targets
the protein.
[0068] Multicolor images obtained from monitoring of random encoded
arrays can be recorded automatically. The arrays can be formed on
chips, and multiple chips can be placed on a carrier, such as that
shown in FIG. 1. However, alternative modes of presenting and
arranging random encoded arrays are possible. For example, samples
also may be mounted in flow cells or cartridges permitting fluidic
operation so as to inject samples, reagents and buffers, permitting
imaging of probe array by way of standard microscope optics.
[0069] The components and subsystems of an exemplary image
acquisition system may include the following:
[0070] Input/Output File System: images are handled in TIFF format,
other files are handled in XML (eXtensible Markup Language) format;
an XML output file records the settings of parameter such as image
acquisition integration time, filter selection System Status:
illumination source (ON/OFF), stage target position Mechanical
Subsystems: xy translator, z actuator, filter wheel,
[0071] ND filters
[0072] Barcode Reader
[0073] Image Acquisition and Storage
[0074] Control Software
[0075] Graphical User Interface (GUI)
[0076] Autocenter Function (implemented by Software)
[0077] Autofocus Function (implemented by Software)
[0078] Hardware. FIG. 4 shows a microscope, for use in an imaging
system, providing transmission or reflection geometry and multiple
methods of generating optical contrast. In one embodiment,
reflection geometry is chosen to record "brightfield" (e.g., an
image not recorded under fluorescence optical contrast imaging
conditions, including an image recorded under white light
illumination) as well as multicolor fluorescence images in a fully
automated manner.
[0079] Other components and subsystems of an imaging system are set
forth below.
[0080] Illumination Source. Depending on the application of
interest, any suitable illumination source (400) can be used,
including a laser, or a standard microscope illumination sources
including tungsten halogen, mercury and xenon. In one embodiment, a
xenon light source is used for multi-fluorescence imaging. A
mechanical shutter (410) controls "Light-ON" and "Light-OFF"
functions.
[0081] Mechanical Subsystems. Certain subsystems can be used to
control precision positioning of the sample (420), selection of
image mode, namely brightfield or (epi)fluorescence (using
different filters), and spectral filter selection. The positioning
of the sample involves horizontal and vertical sample positioning,
deploying a computer-controlled xy-translator (part of the xyz
stage (430)), which can be under the control of a manual "joystick"
positioning function or an automated autocentering function, and a
z-actuator connected to a vertical motion of the sample under
control of an autofocusing function which may be
computer-controlled. Fluorescence filter combinations can be
selected automatically using a computer controlled carousel housing
filter cubes (440).
[0082] Barcode Scanner. A handheld barcode scanner (450) can be
used to scan an identifying barcode affixed to each multichip
carrier or other sample carrier or cartridge. The barcode can
identify, for example, the composition of beads associated with the
carrier, the origin of the carrier (i.e., the batch it is derived
from) or other information.
[0083] Optical Subsystem--A combination of microscope objective
(460) and collection optics (470) in standard configuration, or in
Koehler configuration, is used for illumination as well as
collection and image formation.
[0084] CCD Camera. A CCD camera (480), preferably with C-mount, is
attached to the microscope to record images. Control Software. The
fully automated operation of the array imaging system is enabled by
control software (also referred to herein as the Array Imaging
System--Operation Software ("AIS-OS")) comprising a Graphical User
Interface (GUI) as well as control algorithms implementing
autocentering and autofocus functions, as set forth below.
[0085] Operation of Array Imaging System. Bead arrays mounted in a
multichip carrier or cartridge are placed on the translation stage
of the Array Imaging System ("AIS") and multicolor images (both
decoding and assay images) are recorded, Table I below shows the
pseudocoele for the translation/decoding operation of the Array
Imaging System.
TABLE-US-00001 TABLE I IF (record Decoding Image mode) { LoadFile
(Production Data Record); /** Load or Create **/ ScanBarCode
(Carrier ID); WriteInfo (Carrier ID, Number of BeadChips on
Carrier, ProductionDataRecord); }; ELSE IF (record Assay Image
mode) { ScanBarCode(Carrier ID); ReadInfo (Production Data Record,
CarrierID, Number ofBeadChips on Carrier); /** The Production Data
Record is accessible as an XML file in the folder set up by the AIS
for each sample **/ ConstructFileName (AssayDataRecord) /** to
match Production Data Record **/ WriteInfo(Assay Data Record,
CarrierID, Number of BeadChips on Carrier); /** Production Data
Record and Assay Data Record are maintained on an SQL database
server accessible to authenticated users **/ }; index = 0; WHILE(
index < Number ofBeadChips on Carrier) { IF (record Assay Image
mode) { ReadTargetPosition(Production Data Record, BeadChipID, X,
Y, Z); }; MoveXYTranslator ( X, Y );MoveZTranslator (Z); Z =
AutoFocus(Z); /** perform autofous operation **/ AutoCenter(X, Y);
/** In record Decoding Image mode, operator sets first center
position **/ Z = AutoFocus(Z); /** repeat autofocus **/ IF (record
Decoding Image mode) { WriteTargetPosition(Production Data Record,
BeadChipID, X, Y, Z); }; FOR (each desired ColorChannel) {
SetFilter (ColorChannel); AcquireImage ( ); /** See details below
**/ WriteImageFile(CarrierID, BeadChipID, ColorChannel, ImgFile);
IF (record Decoding Image mode) {
UpdateProductionDataRecord(CarrierID, BeadchipID, ImgFileName); };
ELSE IF (record Assay Image mode) {
UpdateAssayDataRecord(CarrierID, BeadChipID, ImgFileName); }; };
index++ ; };
[0086] Autocentering. The autocentering function, using a given
input image, positions the XY translation stage so as to place each
selected array into the center of the imaging system's field view
by determining that the image in the viewing field is, in fact, a
rectangle with the correct number of sides and right angle corners.
This is accomplished by performing the steps in Table II, which
shows the pseudocode for the autocentering operation:
TABLE-US-00002 TABLE II OpenImg(InputImg, OpenedImg); /** apply
sequence of morphological erosion and dilation operations to
eliminate internal structure of the image showing particle array
**/ Binarize(OpenedImg,BinImg); /** apply optimal thresholding
algorithm **/ CloseImg (BinImg, ClosedImg); /** apply sequence of
morphological dilation and erosion operations **/
AnalyzeConnectivity (ClosedImg, ConComp); /** find connected
components in closed image **/ Filter (ConComp, FilteredConComp);
/** filter out all "non-box-like" regions; a "box-like" region is
defined as a region whose area is close to the area of its
"bounding box" **/ Center = FindMaxConComp(FilteredConComp); /**
find largest connected component that is smaller than 70% of the
image size and find its centroid **/ MoveXYTranslator ( Center.X,
Center.Y ); /** position stage **/
[0087] Only when a new multichip carrier ("MCC") is first inspected
and decoded, does the positioning of the very first array on the
MCC require interactive operation. This initial positioning step is
performed as a part of array assembly or subsequent quality
control. All subsequent positioning may be automatic. The full
processing-positioning followed by acquiring of multiple images and
displaying a rendering of a multicolor image typically requires
only a few minutes.
[0088] Autofocusing. The autofocusing function positions the Z
actuator so as to bring the image in focus and place each selected
array into the center of the imaging system's field view. An
algorithm which uses a local contrast function to determine optimal
focus can be used. This local contrast is determined as follows
using a fast computation: evaluate, for each pixel, a quantity
.DELTA..sub.max, defined as the largest absolute value of the
difference in intensity between that pixel and its four horizontal
and vertical neighbors; next, sum the .DELTA..sub.max over a
designated portion of the image: this serves to speed up the
operation. The Z-position of maximal contrast is located. The
autofocus function should help ensure vertical positioning to
within one micron or less.
I.3 Performing Multi-Analyte Molecular Analysis
[0089] The process of performing multianalyte molecular analysis
using the system herein would, in an exemplary embodiment, involve
the concatenation of the previously described operations as follows
(FIG. 5):
[0090] Collect patient sample (510)
[0091] Transfer to sample container, preferably a barcoded sample
container (520)
[0092] Process sample (530) using requisite reagents (540) to
produce analyte (550)
[0093] Select multichip carrier (MCC), obtain MCC information (560)
(580), for example, positions of beadchips arranged on MCC;
[0094] Perform assay (590) to produce transformed analyte (591) to
be analyzed
[0095] Mount MCC in array imaging system and read MCC barcode (570)
to obtain assay configuration Acquire assay image(s) (592)
[0096] Submit assay image data for processing and analysis, details
of which are described below.
II Image Processing and Analysis
[0097] Processing, analyzing, transmitting and storing images as
set forth herein can be implemented in Visual C++ (MicroSoft) using
a graphical user interface software package including .exe files
implementing the pseudocodes and flow diagram steps described
herein including, for example, the analysis, processing and
decoding steps described herein.
[0098] An image processing program (designated "DECODER"), which
can be run, for example, on the Microsoft Operating system, e.g.,
Windows 98 or 2000, and which contains functions to display,
process, save and print "multicolor" sets of multiple microarray
images in an integrated graphical user interface (GUI) and to
generate a decoding data record which may be submitted for further
analysis to the ANALYZER, residing either on the same computer or
on a separate computer. This program can be readily implemented by
those skilled in the art, using the outlines herein. As illustrated
in FIG. 6, the operation of the DECODER comprises reading the
Decoding Image Record (610, 620), rendering and displaying decoding
image(s) (630), and processing decoding image(s) (640) to create a
Decoding Data Record (650), display scatter plots (660) and update
databases (670).
[0099] Another image processing program (designated READER), which
can be run, for example, on the Microsoft Operating system, e.g.,
Windows 98 or 2000, and functions to display and to process pairs
of assay images acquired so as to generate an assay data record
which may be submitted for further analysis to the ANALYZER,
residing either on the same computer on a separate computer. As
illustrated in FIG. 7, the operation of the READER comprises
reading the Assay Image Record (710, 720), rendering and displaying
assay image(s) (730), and processing assay image(s) (740) to create
an Assay Data Record (750) and update databases (760).
[0100] ANALYZER is an analysis program. As illustrated in FIG. 8,
ANALYZER receives input from DECODER, in the form of a Decoding
Data Record (810), and READER, in the form of an Assay Data Record
(820), and comprises functions to perform further analysis
including: cluster analysis (830) to create a decoding map (840).
The decoding map is combined (850) with an assay data record so as
to produce a Decoded Assay Data Record (860) which is displayed
(870) in a variety of formats and stored in a database (880).
[0101] ANALYZER, DECODER and READER may reside on separate
computers which may communicate by way of a data network. In this
manner, data from assays can be received from a remote site but can
be decoded and analyzed at another site. In such embodiment,
DECODER and ANALYZER may be integrated into a single program or
loaded onto the same computer.
[0102] IChipReader provides a COM interface in the form of a
dynamically linked library linking the functions of DECODER and
READER.
[0103] This image analysis system has the following advantages.
[0104] Reliability: Robust algorithms have been designed in order
to handle images of widely varying quality encountered in practice,
including images exhibiting very low contrast or variations in
contrast across the image, significant noise and corruption of
edges or features, or displacement and misalignments between
multiple images of a given array. These robust algorithms ensure
the reliability of the results produced by the analysis.
[0105] Accuracy: The entire sequence of processing steps is
performed without human intervention, thereby avoiding error and
enhancing ease-of-use.
[0106] Speed: Algorithms have been designed for efficiency, and
functions have been integrated so as to minimize processing time. A
chip displaying a single array can be processed in as little as 4
seconds.
[0107] Productivity and throughput: Sets of images may be analyzed
in batch mode.
[0108] Ease-of-use and convenience: The GUI package provides
convenience and flexibility in controlling all system functions.
Functions and capabilities provided by these systems including
processing, analyzing, transmitting and storing images are
elaborated below.
II.1 Array Segmentation and Extraction of Signal Intensities
[0109] Image processing may be applied to each of the one or more
constituent images of a composite image in the decoding data record
or assay data record to segment the image and extract a textual
representation of the signal intensity distribution within the
array. This representation would serve as input for further
analysis.
[0110] In certain embodiments, decoding and assay images or the
corresponding data records are analyzed to obtain quantitative data
for each bead within an array. The analysis invokes methods and
software implementing such methods to: automatically locate bead
arrays, and beads within arrays, using a bright-field image of the
array as a template; group beads according to type; assign
quantitative intensities to individual beads; reject processing
"blemishes" such as those produced by "matrix" materials of
irregular shape in serum samples; analyze background intensity
statistics; and evaluate the background-corrected mean intensities
for all bead types along with the corresponding variances.
II.1.1 Referenced Arrays
[0111] Referenced arrays are located in designated positions, and
in designated orientations, with respect to features designed into
patterned substrates in accordance with methods previously
disclosed in PCT/US01/20179, U.S. Pat. No. 6,251,691 and U.S.
patent Ser. No. 10/192,352. For example, a locus of low impedance
on a substrate may be designed to collect particles using the
LEAPS.TM. method and may further contain a central recess grid to
mechanically immobilize microparticles. See U.S. Pat. No.
6,251,691.
[0112] Specifically, in one embodiment, following completion of
AutoCentering and AutoFocusing as described above, the system makes
a record, in both the "record Decoding Image" and the "record Assay
Image" modes, of both the brightfield image and one or more color
images of the array, the color images being recorded following
selection of the desired filter settings as described above. In one
embodiment, color images in the "record Decoding Image" mode are
recorded in a "BLUE" and in a "GREEN" channel selected by
respective filter combinations (Blue Channel: excitation filter:
405 nm (20 nm); emission filter: 460 nm (50 nm) and beam splitter:
425 nm (long pass), Green Channel: excitation filter: 480 nm (20
nm); emission filter: 510 nm (20 nm) and beam splitter: 495 nm
(long pass)) and one color image in the "record Assay Image" mode
is recorded in a "RED" channel selected by a filter combination Red
Channel (excitation filter: 640 nm (30 nm); emission filter: 700 nm
(75 nm) and beam splitter: 660 nm (long pass)). The excitation
filters transmit only those wavelengths of the illumination light
that efficiently excite a specific dye, and an emission filter
attenuates all the light transmitted by the excitation filter and
transmits any fluorescence emitted by the specimen, and a beam
splitter reflects the excitation light but transmits the emitted
fluorescence (the figures in parenthesis indicate the width of the
band for each filter). Therefore, the AIS system, in either "record
Decoding Image" mode or "record Assay Image" mode, permits
recording of images in two or more color channels.
[0113] Processing of images recorded from referenced arrays may be
performed by extracting a reference "mesh" or "grid" structure,
where individual fields in the grid include beads and the outer
dimensions of the grid correspond with the dimensions of the
referenced array. The principal operations common to the processing
of Decoding Images and Assay Images include segmentation to locate
array boundaries, mesh/grid delineation, image registration (or
alignment) and extraction of intensities, as elaborated below.
Following completion of processing steps, further analysis is
performed by constructing a decoding map from two or more decoding
images using a cluster algorithm and by merging decoding and assay
data record to produce decoded assay data record. Partial or
complete results and related information may be exchanged between
two or more parties, as further elaborated herein.
[0114] In the "process Decoding Image" mode, the following
operations, as shown in Table III, are utilized. Table III shows
the pseudocode for an exemplary processing of a decoding image.
TABLE-US-00003 TABLE III LoadImage (BrightField Image);
FindBoundary (BrightFieldImage, RotAngle ); /** using brightfield
image, find array boundary and angle of misorientation with respect
to display edges **/ RotateImage(BrightFieldImage, RotAngle);
FindGrid (BrightFieldImage, Grid) /** locus of local intensity
minima **/ FOR (each decoding image be processed) { LoadImage
(Fluorescence Image); RotateImage (Fluorescence Image, RotAngle);
AlignImage(FluorescenceImage); /** align fluorescence image with
bright field image **/ OverlayGrid ( );
ReadIntensityDistrib(DecodingDataRecord, SampleMesh, Grid,
FluorescenceImage); }
[0115] These steps are followed by the step of creating a scatter
plot from two or more decoding images and performing a cluster
analysis to establish a decoding map. These steps are elaborated
below. In the "process Assay Image" mode, the pseudocode for the
processing of an assay image, as illustrated in Table IV, are
used.
TABLE-US-00004 TABLE IV LoadImage (BrightField Image); FindBoundary
(BrightFieldImage, RotAngle ); /** using brightfield image, find
array boundary and angle of misorientation with respect to display
edges **/ RotateImage(BrightFieldImage, RotAngle); FindGrid
(BrightFieldImage, Grid) /** locus of local intensity minima **/
FOR (each assay image to be processed) { LoadImage (AssayImage);
RotateImage (Assay Image, RotAngle); AlignImage(AssayImage); /**
align assay image with bright field image **/ OverlayGrid ( );
ReadIntensityDistrib (AssayDataRecord, SampleMesh, Grid,
AssayImage); }
[0116] Histogram expansion is applied to brightfield and color
images prior to finding boundaries, locating grids and aligning
images; only the intensity extraction is performed on the 16-bit
image as originally recorded. The principal operations are
implemented using standard methods (Seul, O'Gorman & Sammon,
"Practical Algorithms for Image Analysis", Cambridge University
Press) as follows.
[0117] FindBoundary--Array edges in the brightfield image (and
optionally any of the color images) are located using a standard
Sobel y-gradient operator image for left and right edges and a
standard Sobel x-gradient operator for top and bottom edges. Using
these edges, the location of the array and its misorientation with
respect to the image display boundaries are computed. For future
use, the array is rotated to bring it into alignment by with the
image display. Prior to edge detection, noise is filtered by
applying six iterations of a morphological "Open" operation, which
is an image processing technique. See Seul, O'Gorman& Sammon,
supra.
[0118] Mesh/Grid Construction--A grid or mesh delineating intensity
maxima ("peak") is extracted by tracing the locus of local
intensity minima ("valley") within the brightfield image. This
locus defines a mesh or grid such that each field in the mesh
delineates a local intensity maximum associated with a bead or with
a recess provided in patterned substrates. In this manner, the grid
traces around each of the beads, and includes one bead in each
segment of the grid.
[0119] To implement the mesh construction, the problem is mapped to
Dijkstra's "shortest path" algorithm (see Introduction to
Algorithms, T. Cormen, C. Leiserson et al., The MIT Press) well
known in the fields of computational geometry and combinatorial
optimization, by ascertaining the intensities of image pixels with
values of vertices in a graph. The algorithm finds the mesh, also
referred to as a grid, as an optimal path as follows:
Pre-Process Image:
[0120] Compute the external gradient image by subtracting a dilated
image from the original image.
Determine Overall Orientation, Either Horizontal or Vertical
[0121] As illustrated in FIG. 15A, provide horizontal and vertical
reference lines of known length to determine the orientation of the
underlying hexagonal grid, compute shortest paths for the two
reference lines and compare to corresponding reference line lengths
by forming. The ratio closes to unity indicates either horizontal
or vertical orientation.
Find Horizontal Grid Partition:
[0122] Replicate reference lines by shifting by unit mesh size,
then compute shortest path to determine actual grid line. Continue
until replicated line falls outside array boundary.
Find Vertical Grid Partition:
[0123] As illustrated in FIG. 15b, find the shortest path of a
diagonal line--depending on overall orientation--oriented at either
30 degrees or 60 degrees with respect to the horizontal lines in
the case of an anticipated underlying hexagonal lattice of average
intensity maxima. Compute intersections of the diagonal lines and
every horizontal line given the intersections of the diagonal lines
and two consecutive horizontal lines, vertical lines will be
located at the midpoint of these intersections.
Post-Process Grid:
[0124] Grow or shrink grid defined by the totality of the
horizontal and vertical lines to the expected array boundary;
correct grid stagger.
Store Grid:
[0125] Store grid coordinates in a file.
[0126] Image Registration--Given a grid, a registry of the
brightfield image with one or more color images ensures proper
alignment by eliminating possible misorientation and translation
("shifts") between the multiple images recorded from a given array.
One source of such shifts is the wavelength-dependent refraction
introduced by standard fluorescence filter combinations.
Registration aligns the assay and decoding images.
[0127] Misorientation is eliminated by rotation to bring a given
image into alignment with a reference such as the brightfield image
grid. An alternative for aligning images without reference to a
brightfield image is described below.
[0128] Assuming only translational displacement, the system can
invoke the following fast algorithm. To determine horizontal
displacement ("X-shift"), construct intensity profiles along
vertical scan lines within the array boundary; similarly, to
determine vertical displacement ("Y-shift"), construct intensity
profiles along horizontal scan lines within the array boundary.
Next, construct horizontal and vertical profiles along lines
displaced from the first set by one respectively one horizontal or
one vertical mesh unit. The peak in the profile determines the
image shift.
[0129] The methods herein are limited to shifts between images of
less than half of the mesh size, by the optical subsystems of the
system including the CCD camera used herein. If larger shifts are
encountered, image registration may be off by one mesh unit in row
and/or column dimensions. A Minimal Variance Matching algorithm
described below is utilized to correct larger misalignments.
[0130] Intensity Extraction. Following completion of image
registration, in one embodiment, intensities are extracted from
each color image by sampling the interior, and not the exterior, of
each field of the mesh/grid with an averaging filter mask of
suitably chosen size to fit into the interior of each mesh field.
Each intensity value is optionally corrected by subtracting a
supplied background value. One method of supplying the background
values is to record it as an average of pixels values from an area
of the image outside the mesh/grid boundary.
[0131] In contrast to widely used conventional methods that invoke
peak finding and peak fitting algorithms to locate object
positions, the present method offers substantial advantages of
processing speed.
[0132] Extracted intensities are stored--optionally in binary
form--in a one-dimensional array of length L, L denoting the number
of units of the grid/mesh constructed in the course of
segmentation. This one-dimensional array can be mapped onto the
grid or mesh to associate each intensity value with a unique
coordinate within the bead array. For example, the following
structures may be used:
TABLE-US-00005 float intensity[4012]; /** array holding 4012
intensity values **/ Struct Grid{ int leftUpX, int leftUpY, int
rightDownX, int rightDownY}; /** structure representing one grid
field **/ Grid grid[4012]; /** array holding 4012 grid fields
**/
[0133] Eliminating Reference to Brightfield Image. In certain
embodiments, one may eliminate reference to the brightfield image
in the course of image processing, notably during the step of
eliminating image misalignment, and indeed to eliminate the step of
recording the brightfield image altogether. In that case, the step
of aligning color images with the display boundaries invokes
information extracted directly from the color images.
[0134] The approach is conceptually as follows. Given a color
image, construct horizontal and vertical intensity profiles by
respectively projecting image intensities to the top-most and
left-most scan line in the display, then evaluate the intensity
variation in each profile. Next, rotate the color image by a
pre-defined angle and repeat the previous construction. Continue to
rotate until the profiles exhibit maximal variations, then reduce
the step size in rotation angle and reverse the direction of
rotation until the optimal rotation angle is found.
[0135] This procedure is significantly improved when information
about the array geometry is available a priori. For example, in one
embodiment of referenced arrays, a hexagonal geometry with specific
choice of nearest neighbor separation, a, and alignment of
principal axes with the chip edge is chosen. Then, the desired
alignment is characterized by one of the profiles assuming the form
of a periodic variation with a single periodicity, a, and the other
profile assuming the form of a superposition of two phase-shifted
periodic functions, both with periodicity, a*cos 30.degree..
Horizontal and vertical profiles produced by such an array at a
given misalignment angle thus may be analyzed by fitting each to a
superposition of two trial functions and obtaining the angle of
misalignment from the fit.
II1.2 "Non-Referenced" Arrays
[0136] Decoding and Assay Images. To perform a multiplexed binding
assay in accordance with the READ.TM. process, the array is first
imaged by multicolor fluorescence, to determine the color code of
constituent beads which uniquely correspond to the chemical
identity of the probe displayed on the bead surface; second, to
record the fluorescence intensity which indicates the amount of
probe-target complex formed on each bead surface in the course of
the binding or hybridization assay. The process of image detection
and bead decoding is described in PCT/US01/20179 (WO 01/98765),
incorporated herein by reference in its entirety.
[0137] Quantitative Analysis. Image analysis algorithms that are
useful in analyzing the data obtained with the READ process
disclosed herein may be used to obtain quantitative data for each
bead within an array, as set forth in PCT/US01/20179, incorporated
herein by reference. In preferred embodiments, data are obtained
from the decoding and the assay images, or preferably from the
corresponding decoding image record and assay data record by
application of certain algorithms. These algorithm may be used to
obtain quantitative data for each bead within an array. The
analysis software automatically locates bead centers using a
bright-field image of the array as a template, groups beads
according to type, assigns quantitative intensities to individual
beads, rejects "blemishes" such as those produced by "matrix"
materials of irregular shape in serum samples, analyzes background
intensity statistics and evaluates the background-corrected mean
intensities for all bead types along with the corresponding
variances. Using calibration beads that are included in the assay,
intensities are converted to an equivalent number of bead-bound
fluorophores.
II1.2 Representation and Encryption of Array
Configurations--ChipID
[0138] II2.1 Covering. Given a set of probe molecules of types
P={p(1), . . . , p(k), . . . , p(n)} and a set of tags, T={t(1), .
. . , t(k), . . . , t(n)}, the former, for example in the form of
oligonucleotides of defined length and sequence, the latter for
example in the form of color codes associated with a set of beads,
one defines a one-to-one mapping of T onto P whose image represents
a covering, C:=C (P) of the set P. The covering is obtained by
attaching probes in set P to color-encoded beads in set T.
[0139] In certain embodiments, encrypted coverings serve to conceal
the identity of probe molecules associated with tags by revealing,
for each probe molecule, only a label or pointer that is logically
linked to that probe molecule, but not the probe identity itself.
This is disclosed only by a "de-covering" process.
II2.2 Encoding Random Array Configurations
[0140] The random assembly of pooled beads of different types into
a planar array creates a specific configuration, thereby defining a
"random encoding", E, as follows. Given a set of tags, T={t(1), . .
. , t(k), . . . , t(n)}, for example in the form of color codes
associated with a set of pooled particles, define E as the mapping
of T onto a set of positions, V={v(1), . . . , v(l), . . . , v(L)},
constructed as follows: from each of n reservoirs of particles,
each reservoir containing particles that are uniquely associated
with one tag in accordance with T={t(1), . . . , t(k), . . . ,
t(n)}, draw r(k) (indistinguishable) particles and place them into
r(k) positions randomly selected from a set V={v(1), . . ., v(1), .
. ., v(L)}. In a preferred embodiment, V corresponds to the
vertices of a rectangular array, {(i, j); I=1, . . . , I, j=1, . .
. , J} or, equivalently, {1; 1=1, . . . , I*J}, of designated
positions ("traps") in a silicon substrate.
[0141] In certain embodiments, encoding serves as a further level
of encryption to conceal the identity of tags which is revealed
only by the decoding process. In addition, standard encryption
techniques may be applied to further conceal encoding and covering
information. Decoding of the array configuration identifies the tag
assigned to each of the positions within V. For example, each such
color code, identified by a unique tag index, may be obtained by
combining fluorescent dyes of fundamental colors, R, G and B, for
example, in specified ratios to produce beads producing
fluorescence signals of intensities (I.sub.R, I.sub.G, I.sub.B),
for example, in the respective color channels, R, G and B. The
system described herein can maintain this information in a separate
configuration file, which is generated in conjunction with the
production of bead libraries.
[0142] Representations. In one embodiment, encoding is achieved by
creating spectrally distinguishable particles by way of staining
them with two or more dyes in accordance with one of several
possible possibilities. For example, several fluorophore tags in
the form of dyes, Red (R), Green (G) and Blue (B), for example, may
be combined in a variety of fixed R-G-B molar ratios or may be
combined in binary (or other) fashion, each dye being either
present or not present in any given particle type. Decoding of an
array of color-encoded particles is performed as described herein
by recording a set of images showing fluorescence intensities in
separate color channels for each of the fundamental dyes and
determining molar ratios by analyzing intensities in the various
color channels.
[0143] The information from multiple decoding channels may be
represented in a merged decoding intensity array which forms part
of the Decoding Data Record described herein, by listing, for each
position v(1), 1 . . . 1 L, a set of intensities (I.sub.R, I.sub.G,
I.sub.B).sub.1, for example, or listing relative abundances that
are obtained by normalizing intensities by suitable internal
standards. Optionally, to obtain a compact integer representation,
intensities may be represented in binary form, I=2.sup.p,
0.ltoreq.p.ltoreq.16 so that a set of exponents (p.sub.R, p.sub.G,
p.sub.B).sub.1 may be stored for each position.
[0144] Further analysis, performed as described herein in
subsequent sections, serves to construct a decoding map. This map
is composed of clusters, each cluster representing one spectrally
distinguishable particle type which in turn is defined by a triplet
of fundamental tags, such as (I.sub.R, I.sub.G, I.sub.B). Once the
decoding is in hand, clusters may be given a simple index which now
serves as a tag index. That is, the triplet is replaced by a simple
tag index. Accordingly, the random encoded configuration generated
by E may be represented in the form of the random sequence of
L=3r(k) tag indices assigned by the encoding E to positions (v(1),
. . . , v(1), . . . , v(L)). In certain embodiments, it will be
convenient and useful to sort this sequential representation by tag
index so as to obtain a one dimensional array of n lists, the k-th
such list containing the sequence of r(k) array positions occupied
by tag k. If the positions are identified by the corresponding
vertex array index, this provides a particularly compact
representation.
[0145] Alternatively, a representation in the form of a 1-d array
of length L of tag indices also may be convenient. For example, the
configuration of an array composed of 4,096 or 212 beads of 128=27
types, could be stored in 4k*2Bytes=8 kB of non-volatile memory
which could be packaged with the carrier.
[0146] Individual Array Configurations: IntrinsicChipID. In a
preferred embodiment of a random encoded array assembled on a
silicon chip substrate, the array configuration, in any of the
aforementioned representations, provides an identifying tag for the
substrate. See U.S. patent application Ser. No. 10/365,993 "Encoded
Random Arrays and Matrices." filed Feb. 13, 2003, incorporated by
reference. Each such InstrinsicChipID is drawn from the number, S,
of distinguishable configurations of a random encoded array of
I*J=L vertices, given by the number of ways in which n (unordered)
samples of r(k) (indistinguishable) particles of type t(k),
1.ltoreq.k.ltoreq.n, may be distributed among L positions:
S(L; n; r(k), 1.ltoreq.k=n): =L!/r(1)!r(2)!. . . r(k)!. . .
r(n)!
[0147] Illustrating the large number of possible combinations is
the fact that an array of L=16 positions, composed of n=4 bead
types, where each type is represented four times (r(1)=. . .
r(4)=4), can display S(16; 4; r(k)=4;
1.ltoreq.k.ltoreq.4)=16!/(4!){circumflex over (0)}4, or
approximately 63 million configurations. The InstrinsicChipID may
be cast in any of the representations discussed above.
[0148] Degree of Randomness: Autocorrelation Function of Tag
Sequence. Indeed, the degree of randomness of a given bead array is
readily ascertained by constructing the autocorrelation of the tag
sequence corresponding to the random configuration of encoded beads
within the array as elaborated above. For example, a random
sequence of length 22 and composed of three tags, R, G, B with
relative abundance 7/22=1/3, will produce an autocorrelation
function, g, of this type with the following behavior near the
origin:
TABLE-US-00006 Shift: -1 R G G B R G R R B R G R B R G B B R G G B
G g = 3 R G G B R G R R B R G R B R G B B R G G B G Shift: 0 R G G
B R G R R B R G R B R G B B R G G B G g = 22 R G G B R G R R B R G
R B R G B B R G G B G Shift: +1 R G G B R G R R B R G R B R G B B R
G G B G g = 3 R G G B R G R R B R G R B R G B B R G G B G
[0149] Scoring each tag match in the autocorrelation as 1, each tag
mismatch as 0, it is readily seen that the (normalized)
autocorrelation function of the random tag sequence will exhibit a
sharp peak and will drop--within a single unit shift--to the
average value of .about.(1/r){circumflex over (0)}2, r denoting the
average redundancy of each tag. This property of random encoded
arrays will serve to construct a robust "matching by variance
minimization" algorithm to combine decoding and assay data records,
as described herein.
II.3 Image Analysis
[0150] Completion of the aforementioned image processing steps
yields a compact representation of the intensity distributions in
decoding and assay images which facilitates further analysis. This
analysis includes the steps of generating a decoding map from the
set of decoding images and combining ("merging") decoding and assay
images to generate final assay results using a matching algorithm
further elaborated below.
II3.1 Construction of Decoding Map by Cluster Analysis
[0151] A decoding map assigns each bead located in the processing
of a decoding image to a unique group in accordance with its unique
tag. For example, color-encoded beads will be grouped by color
and/or by intensity of each of two or more encoding colors, as
assumed here for clarity in the exposition of the clustering
algorithm. It will be apparent that other codes are possible here
and will be used in analogous fashion. The system herein may
include two or more clustering algorithms.
II3.1.1 Matching to Map Template
[0152] This algorithm anticipates a decoding map template,
constructed manually or otherwise provided, which provides seed
locations, each anticipated group or cluster in the decoding map
corresponding to one such seed. This is particularly advantageous
in the situations commonly encountered in practice involving
analysis of a decoding image recorded from bead arrays of the same
batch or lot. That is, the number of anticipated clusters, and
their respective approximate central locations, are known a priori.
Assuming, for purposes of illustration, ratio encoding by two
encoding colors, the algorithm produces a partition of a given
scatter plot of decoding intensities which is first converted into
a two-dimensional histogram.
[0153] The map template matching algorithm first generates a
two-dimensional histogram image of the input data, optionally
providing smoothing to the histogram image to eliminate noise.
Given the two-dimensional histogram, the decoding group is
generated using a "watershed" algorithm, well known in the art in
connection with image segmentation, which treats the intensity
histogram as a topographical map showing local elevation as
function of position. Starting at the lowest point, the "water
level" is now gradually increased until "water" starts to spread
over two previously separate compartments. A "dam" is constructed
at the "overflow" position. The set of dams so constructed
represents the set of segment boundaries.
[0154] To implement these steps of generating the decoding map, the
map template matching algorithm uses three auxiliary objects: a
priority queue, a stage (of processing) image and a label image.
The priority queue maintains individual pixels in accordance with
their intensity values as obtained from the two-dimensional
histogram of the input scatter plot, keeping the pixel with the
maximal value at the top. The stage image serves to track the stage
of pixel assignment: any given pixel either is or is not assigned
to a group or cluster. The label image serves to track the group
identity of each assigned pixel.
[0155] The algorithm proceeds as follows. For each given seed,
initialize the corresponding pixel in the label image by assigning
it the seed label, add its eight nearest neighbors to the priority
queue and mark each of these pixels "assigned" in the stage image.
Next, pop the top pixel from the queue and inspect its eight
nearest neighbors, ignoring unassigned pixels and checking whether
all "assigned" neighbors have the same label. If so, mark the pixel
with that label, otherwise, leave the pixel unmarked. Finally, add
to the queue all non-zero neighbors not currently in the queue and
pop a new element. Continue until queue is empty--at which point
the label image shows the group assignment for each pixel.
[0156] The resulting partition assigns each data point in the
scatter plot to one and only one of the groups ("clusters")
identified by the set of given seed locations. Two or more of such
scatter plots are processed if three (or more) colors are used for
encoding. The algorithm performs the operations as illustrated in
Table V. Table V shows the pseudocode for the operation of
constructing a Decoding Map by way of template matching.
TABLE-US-00007 TABLE V CreateScatterPlot(Image,
IntensityArray(FirstColor), IntensityArray(SecondColor)); /** 2d
Plot of Intensities extracted from Decoding Images in different
Selected color channels **/
ConvertScatterPlotToTwoDimHistogram(Image); SmoothImage(Image,
Kernel); /** apply 5*5 Smoothing Filter **/
SetMapTemplate(MapTemplate); /** provide map template **/
GetSeedLocations(MapTemplate, ClusterSeeds);
GenerateDecodingMap(ClusterSeeds);
II3.1.2 Fast Clustering Algorithm
[0157] The system described herein also includes a fast algorithm
that invokes graph theory to construct a two-dimensional decoding
map without the aid of a template. The algorithm converts the input
scatter plot of intensities into a "distance graph," each data
point in the scatter plot representing one node in the graph, and
each such node being connected by one edge to its K nearest
neighbors (by Euclidean distance). Each edge is assigned a weight
that is proportional to its length, and each node is given a value
computed from the weight of the largest edge connected to that
node.
[0158] The algorithm comprises the following steps. First, load
scatter plot and convert it to a distance graph image. Process the
graph image by applying a morphological Open operation to each
connected graph--the steps of erosion and dilation constituting the
open operation will alter node values. Next, for each node in turn,
eliminate all edges whose weight exceeds the node's new value.
Then, partition the graph into connected components, a connected
component or cluster being defined as a sub-graph of connected
nodes--each node within a connected subgraph can be visited by
traversing edges. Finally, filter out small groups and split large
groups into two groups if necessary. The algorithm thus performs
the following steps, as illustrated in Table VI. Table VI shows the
pseudocode for the operation of constructing a Decoding Map by fast
cluster analysis.
TABLE-US-00008 TABLE VI CreateScatterPlot(Image,
IntensityArray(FirstColor), IntensityArray(SecondColor));
ConstructDistanceGraph(GraphImage, Image); /** assign value to each
node **/ Open(GraphImage); For each(Node in the GraphImage){ /**
process all nodes in the graph **/ For each(Edge of the Node){ /**
process all edges of a node **/ If( Edge > Node) { DeleteEdge(
); } } } PartitionGraph(GraghImage); /** partition graph into
connected subgraphs ("clusters") **/ PostProcess(GraphImage);
[0159] Three-Color Encoded Objects: "Stacked" Decoding Maps. The
clustering algorithm is applied to handle multi-dimensional cluster
analysis for populations constructed as stacked two-dimensional
clusters by preceding the clustering operation with a sorting step.
In stacked two-dimensional clusters the third decoding image
acquired in a case of encoding by three-color combinations will
have one or more discrete intensity levels. Considering first the
case of just a single intensity level, particles are readily sorted
into two groups, namely those containing the third dye (labeled ON)
and those not containing the third dye (labeled OFF) to obtain a
stack of two-dimensional scatter plots which are individually
analyzed to generate two corresponding decoding maps. In practice,
this operation is performed using the DECODER as follows. First,
generate a two-dimensional scatter plot for the original two dyes,
encoding colors, designated G and B. This "G-B" plot represents an
intermediate result that corresponds to the projection of the
three-dimensional "R-G-B" space onto the "G-B"" plane. To split
this projected scatter plot into its constituent components,
generate a two-dimensional scatter plot just for third color,
designated R, in the "R-R" plane by providing two copies of the
decoding image recorded in the R-channel. The "R-R" plot will have
the same size (and number of eventual clusters) as the "G-B" plot
(containing, after all, images of the very same objects). The "G-B"
plot is now split into two plots, one containing only points
corresponding to "R-OFF", the other containing only points
corresponding to "R-ON". A (two-dimensional) decoding map is now
constructed for each plot using one of the algorithms described
above. This strategy is readily generalized to populations encoded
using multiple levels of a third color.
II.3.2 Combining Assay and Decoding Images
[0160] The identity of the binding agent of the binding
agent-analyte complex is determined by decoding. This step entails
comparison of decoding image(s) and assay image(s). That is, the
assay image is sampled in accordance with the cluster information
in the decoding map to group assay signals by bead type and hence
by encoding tag as described above. A robust matching algorithm
ensuring alignment of decoding images and assay images is described
below.
[0161] Decoding may be carried out at the user site or at a central
location. For example, decoding images--in a suitable
representation such as merged decoding intensity arrays--are made
available to the user, either in the form of text files on a
recording medium that is distributed along with bead arrays or in
the form of a downloadable file available by way of authenticated
access to a central database. Alternatively, decoding may be
carried out on a central server after uploading of the assay image
from the user site. Transaction protocols for this and related mode
of data communication are disclosed below.
[0162] Matching by Variance Minimization (MVM). By construction,
constituent beads of a randomly encoded bead array are randomly
dispersed over the array. Assay signals recorded from a set of
beads randomly drawn from all subpopulations or types of beads,
these beads displaying different types of probes, will exhibit an
inter-population variance, V, that reflects the differences in the
corresponding probe-target molecular interactions. Assuming equal
abundance of all bead types, this inter-population variance may be
approximated by the variance associated with the distribution of
the mean assay signals evaluated over each subpopulation,
namely:
V = j ( I - I j ) 2 , ##EQU00001##
[0163] <I>.sub.j denoting the mean assay signal of the j-th
subpopulation and <<I>> denoting the mean of the
<I>.sub.j. In contrast, assay signals recorded from a set of
beads drawn from the same subpopulation, these beads displaying the
same type of probe, will exhibit an intra-subpopulation variance,
v, that reflects aspects of characteristic remaining chemical
heterogeneities such as bead size, density of probes displayed on
beads, assay binding efficiency, etc, given that all probe-target
interactions within the subpopulation are nominally identical for
each subpopulation; the intra-population variance has the form:
V = k ( I - I k ) 2 , ##EQU00002##
[0164] I.sub.k denoting the assay signal recorded from the k-th
bead within the subpopulation. Except in special circumstances,
assay signals recorded from different subpopulations will be
uncorrelated, and the variance, V, will exceed v:
V>>v
[0165] It is this insight which forms the basis for a robust
"matching by variance minimization" algorithm by which to perform
the cross correlation of decoding and assay images recorded from a
random array and to resolve the task of perfectly aligning the two
(or more) images of interest in the absence of fixed alignment
aides and in the presence of edge-corrupting noise. That is, only
in the correct alignment of decoding image and assay image are
assay signals within the assay image sampled over members of the
same subpopulation. In one embodiment, the alignment is performed
by monitoring the variance, computed for one or more specific
subpopulations of beads that may be included in the array for this
purpose, as the assay image is shifted with respect to the decoding
image.
[0166] As discussed herein in previous section II.2.2, even a
single step displacement will completely scramble the sampling and
mix assay signals from multiple subpopulations. Accordingly, the
correct alignment, even in the presence of considerable edge
corruption, is robustly indicated by minimizing the variance of
assay signals over a subpopulation as a function of relative
displacement. In practice, "dark" particles or objects are filtered
out during this matching step to eliminate erroneous contributions
to the variance. The FilterDarkBeads function is required to
eliminate from the assay image objects or microparticles which do
not contribute a measurable signal because--while the center of
bright objects coincides with the maximum in the intensity profile
it coincides with the minimum in the intensity profile for dark
objects. This can lead to errors in aligning assays and decoding
images. In practice, each array is designed to include one or more
reference subpopulations displaying positive or negative control
probes which are designed to generate a signal of known magnitude
so as to ensure that indeed v <<V. These one or more
reference subpopulations are sampled to minimize the corresponding
subpopulation variances, v, as a function of displacement from
perfect alignment. That is, v is evaluated as the assay image grid
is shifted relative to the decoding image grid. If the condition v
<<V is not satisfied, the algorithm produces a warning to
indicate a possible problem with image quality and provides a
choice to abandon further analysis.
[0167] As illustrated in FIGS. 9A and 9B, there are seven possible
misaligned positions if the search is confined to single row and
single column misalignments, namely: (-1, -1), (-1, 1), (-1, 0),
(0, 0), (1, 0), (-1, 1), (1, 1); that is, to account for the
stagger introduced in the image grid derived from a hexagonal
lattice (910, 920), seven distinct positions are checked, as
illustrated in FIG. 9C.
[0168] The Minimal Variance Matching algorithm operates on Decoding
and Assay Data Records and evaluates the desired variance as a
function of unit shifts applied to the two images represented in
the respective data records. Table VII shows the pseudocode for the
operation of combining decoding and assay images by way of Matching
by Variance Minimization.
TABLE-US-00009 TABLE VII LoadDecodingData(DecodingDataRecord,
DecodingMap); LoadAssayData(AssayDataRecord, IntensityArray);
MinVariance = -1000; MinVarianceLocation = 0; /** Check Variance
Produced by 7 Possible Unit Displacements - see Text **/ For (i=0;
i< 7; i++){ ShiftGridPosition(i); FilterDarkBeads( );
Variance[i] = Merge(DecodingMap, IntensityArray); IF(Variance[i]
< MinVariance){ MinVariance = Variance [i]; MinVarianceLocation
= i; } }; WriteAssayData(MinVarianceLocation, AssayDataRecord); /**
Save Location **/
[0169] Correction for Multiple Scattering Effects. Unless suspended
in specially selected density matching fluids which generally will
be incompatible with bioanalytical assays of interest, polymeric,
ceramic or other microparticles will exhibit strong scattering of
visible light so that multiple scattering effects are readily
observed in planar assemblies and arrays of such particles. For
example, fluorescence emitted by a microparticle within such a
close-packed planar array is diffracted by its nearest neighbors
and possibly its more distant neighbors, a source of potential
error in overestimating assay signals. This effect is strongly
dependent on interparticle distance and may be diminished or
eliminated by appropriate array and substrate design as well as
choice of illumination and collection optics.
[0170] In addition, the system herein also offers a method of
correcting for the effects of multiple scattering on the intensity
distribution recorded from subpopulations of beads within a planar
array. As with the MVM algorithm, this method takes advantage of
the random spatial distribution of different bead types throughput
the array. Randomly placed "blank" beads, drawn from a
subpopulation that is at most weakly fluorescent for purposes of
encoding, serve as local "antennae" to establish a random sample of
excess fluorescence produced by way of diffraction by the nearest
neighbor configurations encountered within the array. To correct
for the principal global effect of multiple scattering on assay
signals recorded from a random encoded array, the variance of this
excess fluorescence signal is subtracted from the intra-population
variance of all subpopulations.
III Transmission of Image Data Records
III.1 Identifying and Tracking Bead Arrays
[0171] As described in U.S. patent application Ser. Nos.
10/238,439, and 10/365,993, entitled: "Encoded Random Arrays and
Matrices" (the specification corresponding to WO 01/20593),
incorporated herein by reference, and as further elaborated herein
in Sections II2.1 and II2.2, each bead array generates its own
unique ID ("Intrinsic ChipID" or "ChipID") and can be identified.
This ChipID may be physically or logically linked to a CarrierID.
For example, multiple bead arrays may be mounted on a multichip
carrier comprising a bar code which is capable of recording the
identity of each of the bead arrays. The CarrierID is tracked in
the course of producing bead arrays and also is tracked in the
course of acquiring, processing and analyzing assay images using
the system of the invention. The IntxinsicChipID may be linked to a
CarrierID, and to a further assigned ChipID which may be appended
to the CarrierID ("Appended ChipID") or may be otherwise physically
linked to the CarrierID. Unless specifically indicated below, the
ChipID shall be understood to refer to Intrinsic ChipID or Appended
ChipID.
[0172] Samples of interest for chemical analysis including samples
collected from patients for clinical or other testing, also may be
given a sample or patient ID, e.g., in the form of a barcode which
may be tracked along with the CarrierID using a barcode scanner. In
addition, methods of intra-analyte molecular labeling have been
previously disclosed. For these purposes, the addition of unique
molecular external labels or internal labels, for example in the
form of a DNA fingerprints, may be considered. Such labels and
associated methods have been described in U.S. patent Ser. No.
10/238,439. Information derived from the examination of these
molecular labels may be entered into decoding and/or assay image
records by the system herein to minimize sample handling error and
to facilitate the secure exchange of information, as elaborated
herein below.
III.2 Transmission Protocols
[0173] In one embodiment, an assay image may be submitted for
analysis by transmission over a network connection to a central
location. Software available on centrally located servers, in one
embodiment involving the ANALYZER described herein, completes the
analysis and makes results of the analysis available to authorized
users for retrieval. The analysis-server model provides protocols
governing exchange of information in one or more two party
transactions between one or more users and a central server where
data is analyzed (FIG. 10a), and further provides protocols
governing the exchange of information in three-party transactions,
involving patient, to provider to a testing center where data is
analyzed and assay results are recorded (FIG. 10b). A three-party
transaction may also involve a recipient, a mediator and a provider
of information.
[0174] This model offers several advantages to users as well as
suppliers of molecular diagnostics, particularly when
instrumentation distributed to field locations is easy to use and
maintain, while the analysis of the data obtained using the
instruments is complex. To the users' benefit, the requirement for
designated staff with the training and expertise to install, master
and operate analytical software is eliminated. Rapid turn-around of
even advanced data analysis is ensured by access to rapid network
connections even in remote locations at a doctor's office or
patient site. Suppliers benefit from the reduction in cost
associated with the logistics of providing extensive technical
software support while providing high-speed analysis on dedicated
server hardware. In general, the analysis service provider,
equipment manufacturer and assay developer all may be distinct
parties. Additional parties may participate in certain
transactions. For example, the manufacturer of the chips and
arrays, and the analysis service provider, may not be the same
party.
[0175] The system herein provides for an analysis server model
invoking transaction protocols such as those elaborated below that
ensure the secure exchange of private information created, for
example, in genetic analysis--an issue of wide concern. Additional
services, such as advanced analysis in the form of binding pattern
matching via database searches or data archiving, are readily
integrated. In one embodiment, the analysis server model applies to
assays producing data in the form of images and the analysis of
interest relates to the analysis and archiving of images.
[0176] In one embodiment of such a transaction, the exchange of
information may relate to the completion of analytical chemical,
biochemical and diagnostic test, and the participating recipient,
intermediary and provider of the information of interest (e.g.,
personal genetic infoimation) may correspond to patient, testing
center and (data) analysis provider, respectively. Protocols are
set forth for the secure creation, exchange and storage of
information
Transaction Protocols
[0177] Pattern Matching via Access to "Fingerprint" Database. A
pharmaceutical company researcher ("CLIENT") submits data recorded
from an assay performed in an array format to probe the interaction
between a set of immobilized proteins ("receptors") and a second
set of proteins or ligands provided in a solution that is brought
in contact with the receptor array. The data may be in the form of
a decoded assay data record as described herein.
Intensities--recorded from bead arrays or from other arrays,
including "spotted" arrays--reflect a certain pattern of
interactions between receptors and ligands. Alternatively, the data
may represent a pattern of expression levels for a set of
designated genes of interest that may indicate an individual
patient's response to treatment or may indicate a toxicology
profile or may indicate the response triggered by a compound of
interest that is to mimic the action of a known drug, said action
being characterized on a molecular level by said expression
pattern.
[0178] The two-party transaction between PROVIDER and CLIENT is
performed in accordance with a protocol that preserves the
anonymity of the CLIENT and simply permits the CLIENT to search a
PROVIDER database for matching the interaction pattern or
expression pattern with a unique pattern (or "fingerprint").
[0179] Advanced Services. Once decoded assay results are in hand,
additional analysis may be optionally performed. In the simplest
instance, statistical measures such as mean or variance are readily
evaluated over each of the subsets. More generally, the presence of
characteristic "patterns" of receptor-ligand interaction may be
ascertained Such patterns may be indexed and stored in a searchable
database to provide the basis for assay interpretation. This in
turn will facilitate tracking and interpretation of disease
histories and clinical trial results and aid in the identification
of molecular identifiers and features ("genotype") associated with
clinical pathology ("phenotype").
[0180] File Serving Authenticated Remote Access to Decoding Image.
Decryption of the message contained within the assay image by
application of the key represented by the decoding image groups
intensities in accordance with bead type is provided. Following
bead array assembly, the decoding image is analyzed to derive a
temporary ChipID representing a portion of the complete ChipID,
based, for example, on the first row or column of the decoding
image. The ChipID is stored, the temporary ChipID is transmitted to
the user as a password for access to the database and retrieval of
the full decoding image. In certain situations, it may be
advantageous to the user not to download the full decoding image.
For example, if assay results are negative, conclusions about a set
of tested receptor-ligand interactions may be reached without
decryption.
[0181] Alternatively, assay images may be uploaded to the server
for additional analysis or archiving. Incoming assay images axe
linked to stored decoding images by matching temporary and full
ChipID codes.
[0182] In this file server model, fees may be charged in accordance
with the volume of transactions on a single transaction basis or on
a subscription basis in accordance with pricing models practiced in
the application server market.
A Two-Party Transaction Relating to the Analysis of Encrypted Assay
Data.
[0183] The following two-party transaction (FIG. 10a) illustrated
here using an embodiment in the form of custom bead arrays, invokes
an encrypted covering to ensure that the identity of compounds in
the assay'remain private.
[0184] For example, a pharmaceutical company researcher ("CLIENT")
provides to the custom bead array provider who, in this example,
also is the analysis service provider ("PROVIDER"), a library of
compounds to be subjected to on-chip assays in labeled containers
with instructions to create an encrypted covering by simply
recording the container labels corresponding to specific bead
types. For example, "compound in container labeled A anchored to
bead tag T1." The identity of compounds within labeled containers
is known only to CLIENT. In a preferred embodiment, a unique set of
bead tags is selected for a specific client to minimize mishandling
or inadvertent swapping of compound libraries.
[0185] The two-party transaction between PROVIDER and CLIENT is
performed in accordance with the following protocol:
Provider--Provide Bead Array with Unique ChipID
[0186] Create encrypted covering by attaching designated compounds
to "tagged" beads
[0187] Create array encoding by assembling pooled beads into an
array
[0188] Decode array configuration
[0189] Establish ChipID. In a preferred embodiment, the ChipID is
derived from the array configuration
[0190] Optionally, store ChipID in non-volatile memory to be
packaged along with bead array chip Create a public database record
("Key") of the form (ChipID, Encrypted Covering)
[0191] Send bead array chip in assay cartridge (with ChipID) to
Client
[0192] CLIENT--Perform Assay and Transmit Assay Image
[0193] Receive assay cartridge from Provider
[0194] Place analyte solution into assay cartridge and perform
assay
[0195] Record ChipID. In one embodiment, use a chip carrier
containing ChipID in electronic representation in conjunction with
an electronic reader and the array imaging system such that the
ChipID, read out from the assay chip, is recorded and stored, and
thus unambiguously linked, with the encrypted message in a public
record (ChipID/Public, Encrypted Message/Public) Record assay image
and create assay data record ("Encrypted Message")
[0196] Send combination of ChipID and Encrypted Message to PROVIDER
for analysis
Provider--Perform Image/Data Analysis
[0197] Receive public record (ChipID, Encrypted Message) from
CLIENT
[0198] Strip ChipID and check database for matching record (ChipID,
Encrypted Covering)
[0199] Use ChipID to decode message, thereby creating a profile. In
one embodiment, the profile representing the decoded message has
the form {<I>.sub.k, <k <.ltoreq.n}, where the
<I>.sub.k represent intensities averaged over all beads of
tag type k, tags being uniquely associated with a specific compound
in accordance with the encrypted covering
[0200] Create updated database record (ChipID, Encrypted Covering,
Profile)
Provider--Transmit Profile
[0201] Supply database record (ChipID, Encrypted Covering, Profile)
for retrieval by CLIENT.
[0202] A Three-Party Transaction for the Secure Exchange of Genetic
Information. By generating, either concurrently with the completion
of genetic analysis or by concurrent analysis of tagging molecules
added to patient samples, a molecular ED such as a DNA fingerprint
(as described in U.S. patent application Ser. No. 10/238,439) that
is embedded within the assay image, the methods of the present
invention create an unambiguous link between a chip ID
("IntrinsicChipID") derived from the configuration of a random
encoded bead array and a unique genetic ID, thereby not only
minimizing the possibility of error in sample handling but also
enabling verification of assay results and securing confidential
genetic information in the course of two-party or multi-party
transactions, as elaborated below for a three-party
transaction.
[0203] The process illustrated in FIG. 10C using a preferred
embodiment in the form of custom bead arrays packaged on a carrier
within a fluidic assay cartridge ensures the confidentiality of
personal genetic information. Specifically, a three-party
transaction between custom bead array provider who, in this
example, also is the analysis service provider ("PROVIDER"), an
intermediary or facilitator such as an assay, service provider
("TESTING CENTER") and user ("PATIENT") may be organized as follows
to ensure privacy of information generated by genetic testing.
[0204] In this protocol, transactions between PROVIDER and TESTING
CENTER and between TESTING CENTER and PATIENT involve public
records that are identified by the ChipID. A separate transaction
between PROVIDER and PATIENT involves the private information in
the form of the genetic profile. The protocol below ensures that
the identity of the PATIENT is concealed from PROVIDER: the PATIENT
is identified only by the genetic ID presented for authentication
in the final retrieval of genetic information. On the other hand,
in the protocol below, genetic information is made available--by
way of a "Relay" step--to the TESTING CENTER--or a designated
physician or genetic counselor--for communication to the PATIENT.
The three-party transaction (FIG. 10C) between PROVIDER, TESTING
CENTER and PATIENT is carried out in accordance with the following
protocol.
Provider--Provide Encoded Bead Array Chip with Unique ChipID
[0205] Create covering by attaching probe molecules to
color-encoded beads
[0206] Create array encoding by assembling pooled beads into an
array
[0207] Decode configuration to establish ChipID
[0208] Optionally, store ChipID in non-volatile memory to be
packaged along with bead array chip
[0209] Create a database record ("Key") of the form (ChipID/Public,
Covering/Private)
[0210] Send packaged chip (with ChipID) to TESTING CENTER
PATIENT--Request Analysis
[0211] Submit sample to TESTING CENTER
[0212] Receive ChipID from TESTING CENTER
TESTING CENTER--Perform Assay
[0213] Receive assay cartridge from PROVIDER
[0214] Collect sample from PATIENT into assay cartridge
[0215] Send ChipID to PATIENT [0216] Complete sample preparation
and perform genetic analysis
TESTING CENTER--Record Assay Image and Transmit Assay Data
Record
[0217] Record assay image with embedded GeneticID ("Encrypted
Message")
[0218] Send combination of ChipID and Encrypted Message to PROVIDER
for analysis. In a preferred embodiment, use a chip carrier
containing ChipID in electronic representation in conjunction with
an electronic reader and an image acquisition system under general
processor control such that the ChipID, read out from the assay
chip, is recorded and stored, and thus unambiguously linked, with
the encrypted message in a public record (ChipID/Public, Encrypted
Message/Public)
[0219] For later verification: store assay cartridge, optionally
containing patient blood sample
Provider--Perform Image/Data Analysis
[0220] Receive public record (ChipiD/Public, Encrypted
Message/Public) from TESTING CENTER
[0221] Strip ChipID and check database for matching decoding data
record (ChipID/Public, Covering/Private)
[0222] Use covering to fully decode message identifying genetic
profile and embedded GeneticlD
[0223] In one embodiment, the decoded message has the form of
intensities, {<I>.sub.k, 1.ltoreq.k.ltoreq.n}, averaged over
beads of the same type, each type uniquely identifying a specific
probe molecule; other representations also are available as
discussed herein.
[0224] Extract GeneticlD from decoded assay data record image as
the subset of intensities <I>.sub.k corresponding to
ID-specific probes
[0225] Create updated database record (ChipID/Public,
Covering/Private, GenID/Public, GenProfile/Private)
Provider--Transmit Genetic ID
[0226] Send (ChipID/Public, GenID/Public) to Testing Center for
transmission to Patient
PATIENT--Receive Genetic ID and Retrieve Genetic Profile (1080)
[0227] Using (ChipID/Public, GenID/Public), query Provider
database
Provider--Transmit Genetic Profile (1090)
[0228] Authenticate GenID to authorize retrieval of private genetic
profile from database If and only if, authentication confirmed,
retrieve (ChipID/Public, Covering/Private, GenID/Public,
GenProfile/Private)
[0229] Supply database record (GenID/Public, GenProfile/Private)
for retrieval by PATIENT
[0230] Using an assay cartridge, a physical linkage can be created
between patient sample, assay cartridge and BeadChip with
associated ChipID while the embedded genetic ID creates a physical
linkage between genetic identity and genetic profile as an inherent
part of the assay. Verification is then always possible by
retesting. The physical and logical linkages created by the methods
of the present invention between patient sample, ChipID and genetic
profile with embedded genetic ID eliminate common sources of error
in genetic testing such as switching of patient samples.
[0231] Other transaction protocols may be devised using data
structures of the type introduced in the foregoing example, to
ensure that only the PATIENT has access to genetic (or other)
information created in an assay performed at the TESTING CENTER.
For example, in one embodiment of the present invention, the
PATIENT already may be in possession of his/her GeneticID prior to
initiating a three-party transaction. In that case, the steps of
transmitting, relaying and receiving GeneticID (FIGS. 10c, 1050,
1060, and 1070) may be eliminated. Instead, the PATIENT directly
requests transmission of the genetic profile from the
PROVIDER--access to the relevant database may be authenticated by
comparing the Genetic ID used in the request with the Genetic ID
extracted from the genetic profile.
[0232] Decoded assay data records may be archived. Archived decoded
assay data records would be accessed only by those in possession of
a GeneticID or equivalent key embedded in the decoded assay data
record. That is, the database of archived records would be searched
by rapid cross-correlation with the authentication code.
[0233] More generally, the following three-party protocol ensures
that only the PATIENT (or his/her designee, such as a physician)
who initiates a testing procedure is in possession of private
information created in the test performed at the TESTING CENTER and
analyzed by the application service PROVIDER. The TESTING CENTER
has no access to the private information and PROVIDER has no
knowledge of the identity or particulars of the PATIENT. The
PATIENT, having requested and having been assigned, a ChipID and
SamplelD, requests, directly from the PROVIDER, a confidential
authentication key. In one embodiment, this is accomplished by
access to the PROVIDER site, for example by a remote login. If the
confidential authentication key generated by the combination of
ChipID and SampleID is taken by a third party, the PATIENT will
have immediate knowledge that the confidential authentication key
may be at risk of disclosure to a third party. The PATIENT may be
able to request a new SamplelD before providing a new biological
sample to the TESTING CENTER. Software then assigns a randomly
selected encrypted personalized authentication key to the
combination of ChipID/SampleID presented in the request. Only one
such assignment is permitted. In one embodiment, the encrypted
authentication key has the form of a "cookie" that is placed in a
hidden directory on the hard drive or other storage device of the
requesting machine so that only that machine is authenticated for
future retrieval of testing data from the PROVIDER. The PATIENT
will ensure the integrity of the process: should an unauthorized
party, for example, at the TESTING CENTER, attempt to acquire an
authentication key, the subsequent attempt by the PATIENT to do so
would fail, alarming the PATIENT to a possible breach in protocol.
In one embodiment, the encrypted authentication key assigned to the
requesting ChipID/Sample ID combination will be the
IntrinsicChipID, or information embedded therein. In one
embodiment, the random string of integers indicating vertex
positions of a designated specific bead type within the BeadChip
may be used. Following submission by the TESTING CENTER of the
Assay Data Record, identified by a ChipID, for analysis, the
PROVIDER combines the Assay Data Record with the Decoding Data
Record corresponding to the submitted ChipID so as to create a
decoded Assay Data Record from which specific embedded information
such as a genetic profile may be extracted by "De-Covering", that
is, application of the Covering to identify specific probes within
the array as previously elaborated herein. This information is made
available for retrieval by the PATIENT using the encrypted
authentication key previously assigned to the ChipID/Sample ID
combination. In one embodiment, only the machine previously endowed
with a "cookie" will be permitted to access the database containing
the requested information. This protocol ensures that the TESTING
CENTER knows only the identity of the PATIENT but not the
information such as a genetic profile extracted from the assay
while the PROVIDER knows the information such as a genetic profile
but not the identity of the PATIENT.
[0234] It will be apparent to those skilled in the art that the
foregoing specific instances of two-party and three-party
transactions merely illustrate the concepts involved which are
applicable to a wider range of applications.
[0235] Pricing Strategies. The analysis server model of the present
invention provides "fee-for-service"--in a single transaction
format or in subscription pricing format--in which the initial cost
of instrumentation as well as the recurring cost for disposable
items can be absorbed in the charges for one or more of a palette
of services. This has the advantage of eliminating user capital
expenditures. The charges are for analysis, not for enabling
instrumentation or assay components.
EXAMPLES
Example 1
Acquiring and Processing Decoding Image(s)
[0236] FIG. 11 illustrates the processing steps performed by the
DECODER. Displayed in a Graphical User Interface (GUI) (1100) are
three images, namely: a Brightfield image (1110), a green
fluorescence image (1120) and a blue fluorescence image (1130).
Grids, extracted and aligned by the DECODER, also are displayed.
Also displayed is a scatter plot (1140) produced by the DECODER
from the intensities in green and blue fluorescence images.
Example 2
Constructing Decoding Map
[0237] FIG. 12 illustrates the construction of the 2D decoding map
by the ANALYZER which may be integrated with the DECODER whose GUI
is shown (1200). The map of the present example is composed of 33
clusters (1210), each of which is assigned a unique tag index. This
is displayed for each cluster along with the number of beads
contained in that cluster. For example, cluster 1 contains 101
beads.
Example 3
Acquiring and Processing Assay Image(s)
[0238] FIG. 13 illustrates the processing steps performed by the
READER. Displayed in a GUI (1300) are three images, namely: a
Brightfield image (1310), a fluorescence image (1320). Grids,
extracted and aligned by the READER, also are displayed.
Example 4
Analyzing Images and Extracting Representations
[0239] FIG. 14 illustrates the Decoded Assay Data Record in two
sections, namely: a text display (1400) listing assay signals
extracted from the assay data record along with tag indices
assigning each signal to a cluster and hence to a color code; and,
a bar graph display (1420) of data from the Decoded Assay Data
Record.
[0240] It should be understood that the foregoing examples and
descriptions are exemplary only and not limiting, and that all
methods and processes set forth are not to be limited to any
particular order or sequence, unless specified, and that the scope
of the invention is defined only in the claims which follow, and
includes all equivalents of the claims.
* * * * *