Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND APPARATUS FOR BLOOD TYPING
WITH OPTICAL BIO-DISCS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. Patent Application Serial
No.
09/988,850 filed November 19, 2001, which claims the benefit of priority from
U.S.
Provisional Application No. 60/249,477 filed November 17, 2000 and U.S.
Provisional
Application No. 60/252,726 filed November 22, 2000.
The present application also claims the benefit of priority from U.S.
Provisional
Application No. 60/353,014 filed January 29, 2002; U.S. Provisional
Application No.
60/353,773 filed January 31, 2002; U.S. Provisional Application No. 60/375,568
filed
April 25, 2002; and U.S. Provisional Application No. 60/379,045 filed May 9,
2002. All
of the above applications are hereby incorporated herein by reference in their
entireties.
FIELD OF THE INVENTION
The present invention relates to the field of diagnostic assays and biological
analysis and to identification of cell types and antibodies present in a
biological
sample and analyses related thereto. The invention is further related to the
manufacture and use of optically readable discs for biological analysis.
BACKGROUND OF THE INVENTION
Medical diagnostic assays are critical to the diagnosis and treatment of
disease, as well as the general maintenance of good health. Particularly
useful are
the biological and chemical assays performed on whole blood or its components.
One early area of development in the field is related to blood typing for the
purposes
of transfusion. In 1901 Karl Landsteiner discovered that when the blood of one
human being was transfused with that of another human being, differences in
their
blood might well be the cause of shock, jaundice, and the blood disorder
hemoglobinuria that had resulted through earlier blood transfusions.
Landsteiner
classified human blood into A, B, and O groups and demonstrated that
transfusions
between humans of the same blood group did not result in the destruction of
new
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blood cells and that this catastrophe occurred only when a person was
transfused with
the blood of a person belonging to a different group. A fourth main blood
type, AB
was found in 1902 by A. Decastrello and A. Sturli.
From that time, differing blood typing systems have been devised. Historically
the naming of blood grouping systems has been disorganized. The common
conventions stipulating that dominant traits are given capital letters and
recessive
traits are designated with lower case letters have not been followed. Also by
tradition,
red cell antigens were given alphabetical designations or were named after the
family
of the antibody producer.
The International Society of Blood Transfusion (ISBT) (National Blood
Service/Lancaster, PO Box 111, Royal Lancaster Infirmary, Ashton Road,
Lancaster
LA1 4GT, England) has instituted a numerical system of nomenclature to help
standardize red cell blood group terminology. This convention mandates that
each
system and collection has been given a number and letter designation, and each
antigen within the system is numbered sequentially in order of discovery. As
of this
writing, over 20 blood group systems and seven antigen collections have been
defined.
The structure of the antigen determinants for the ABO blood typing system was
established in the 1950s by Watkins and Morgan (Nature 180:1038-1040, 1957),
and
Kabat et al. (Blood Group Substrates: Their Chemistry and Immuno-Chemistry,
1956,
Academics Press, New York). Numerous sera and isolated antibodies have been
used for ABO blood typing purposes. For example, United States Patent No.
4,764,465 to Foung et al. (1988) entitled "Human Monoclonal Antibody Against
Group
A Red Blood Cells" is directed to a human monoclonal antibody that directly
agglutinates type A human red blood cells. The exemplified antibody is an IgM
and is
produced by hybrid cells lines S-H22 and HHA1.
More recently, genes encoding the antigenic determinants have also been
identified. See for example United States Patent No. 5,326,857 to Yamamoto et
al.
(1994) entitled "ABO Genotyping" which discloses genes defining the ABO histo-
blood
groups and methods for the identification of histo-blood group ABO status. The
methods described include the use of DNA probes or size separation of DNA
fragments unique to a blood group status, DNA constructs, recombinant methods
for
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providing histo-blood glycosyltransferases, methods for tumor suppression,
purified
histo-blood group glycosyltransferases, and antibodies produced therefrom
which bind
to protein epitopes.
A variety of~ apparatuses have been utilized to perform ABO blood typing
analysis. For example, United States Patent No. 4,650,662 to Goldfinger et al.
(1987)
entitled "Portable Blood Typing Apparatus and Method" discloses a portable
apparatus to enable rapid determination of an individual's ABO blood group and
Rh
blood type and a method of using such apparatus. The apparatus has a plurality
of
microtubes joined together that contain blood taken from an individual. The
assembly
of microtubes is connected during use to an assembly of reaction chambers
containing blood typing reagents. The apparatus enables rapid visualization of
the test
reactions within the reaction chambers, and may be used in locations removed
from a
laboratory to determine the ABO blood group and Rh blood type of an
individual.
United States Patent 5,324,479 to Naldoni et al. (1994) entitled "Analyzer for
the Determination of the Phenotype and the ABO Blood Group" discloses an
analyzer
for the determination of the ABO blood type of a patient. The analyzer
comprises a
rotatable plate carrying sample-bearing test-tubes and dilution test-tubes
arranged
along concentric circumferences; a dispensing needle which is movable by
mechanical means between a washing position, a position for drawing a sample,
a
position for diluting the sample and a position for introducing the sample
into a
reading well; a station for washing said needle; a conveyor unit for conveying
carrier
members which are provided with twelve reaction wells to a position for
receiving
diluted or the undiluted samples from the dispensing needle; an automatic
feeder that
feeds small balls into each of the wells during the forward motion along the
conveyor
unit; mechanical means for transferring the carrier member to a reading zone;
a unit
that meters the specific antiserum or red cells into each one of the wells;
and an
optical reading device that horizontally reads the transmittance of each one
of the
wells, starting from the moment when antiserum or red cells are introduced;
and a
processor for functionally controlling the analyzer and for issuing an
estimate of the
results of the analyses.
U.S. Patent No. 6,030,581 entitled "Laboratory In A Disc" describes an
apparatus that includes an optical disc, having a substantially , self-
contained assay
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means for binding an analyte suspected of being in a sample. U.S. Patent No.
5,892,577 entitled "Apparatus and Method for Carrying Out Analysis of Samples"
describes systems and methods for conducting an optical inspection of a
biological,
chemical or biochemical sample supported by an optical transparent disc.
U.S. Patent No. 6,143,510, entitled "Measuring Method Using Whole Blood
Sample" describes methods for quantitatively measuring analytes in an
undiluted
whole blood sample by contacting the sample with magnetic particles coated
with a
binding partner, which binds to an analyte in the sample. There is no
description of
this assay being carried out on an optical bio-disc. In addition, U.S. Patent
No.
5,993,665, entitled "Quantitative Cell Analysis Methods Employing Magnetic
Separation" describes immobilization of microscopic entities into a defined
region in a
collection chamber such that analysis by automated means is possible. The '665
patent describes quantitative collection of magnetically labeled target
entities.
There remains a need in the art of medical diagnostics for more efficient and
less expensive diagnostic techniques. As compared to prior methods and
systems,
we have developed a simple, miniaturized, ultra-sensitive, inexpensive system
for
imaging and analyzing cells and their components. This system uses optical bio-
discs, related detection assemblies, as well as information and signal
processing
methods and software.
SUMMARY OF THE INVENTION
In a first aspect, the invention provides a method for determining a blood
group
type of an individual by direct typing on an optical bio-disc comprising
applying red
blood cells to at least one chamber, channel, microfluidic channel, or micro-
channel in
an optical bio-disc, the chamber surface including at least one capture field
including
a capture antibody, at least one positive control field, and at least one
negative control
field; incubating the samples in the disc to promote antigen-antibody
interaction;
placing the disc into an optical reader that supports it on a first side;
rotating the disc
about an axis substantially perpendicular to the first side to separate non-
captured
cells from captured cells located on the chamber surface; obtaining a
measurement
for the test field, the positive control field, and the negative control field
analyzing the
measurement of the test field, the positive control field and the negative
control field
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to determine blood group type of the individual. In certain embodiments of the
first
aspect, the capture antibody is an IgG antibody or the capture antibody is an
IgM
antibody.
In certain embodiments of the first aspect, the capture antibody is an
antibody
specific for a red blood cell antigen. In certain embodiments of this
embodiment, the
red blood cell antigen is an ABO system blood group antigen, the red blood
cell
antigen is an Rh system blood group antigen, the red blood cell antigen is an
MNSs
system blood group antigen, the red blood cell antigen is a P system blood
group
antigen, the red blood cell antigen is a Lutheran system blood group antigen,
the red
blood cell antigen is a Kell system blood group antigen, the red blood cell
antigen is a
Lewis system blood group antigen, the red blood cell antigen is a Duffy system
blood
group antigen, the red blood cell antigen is a Kidd system blood group
antigen, the
red blood cell antigen is a Fisher system blood group antigen, or the red
blood cell
antigen is a blood group antigen from any other blood group.
In other certain embodiments, the optical bio-disc is a reflective disc or the
optical bio-disc is a transmissive disc. In other embodiments of the first
aspect, the
optical bio-disc comprises a CD, CD-R, DVD, or a DVD-R.
In certain embodiments of this aspect, the optical bio-disc has software
embedded therein and the analysis of the measurement profile is controlled by
the
software, resulting in a bar code displaying the blood group type of the
individual.
In certain embodiments of the first aspect, the capture antibody is
biotinylated
and is bound to the test field by streptavidin bound thereto, or the capture
antibody is
bound to the test field by a second antibody bound to the test field, or the
capture
antibody is bound to a second antibody which is biotinylated and is bound to
the test
field by streptavidin bound thereto.
In other embodiments of the first aspect, the positive control field has a
molecule on its surface that binds all cells. In embodiments thereof, the
molecule is a
lectin. In another embodiment thereof, the molecule is gold.
In a second aspect, the invention provides, a method for determining the
presence of antibodies to an ABO blood group in individual's blood sample by
reverse-typing on an optical bio-disc including purifying serum from a blood
sample;
creating at least one sample by mixing serum with cells of a known ABO blood
group;
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injecting at least one sample into at least one channel in the optical bio-
disc, thereby
delivering the sample onto a capture field including a cell binding molecule;
incubating
the sample on the capture field to allow the cells to bind to the cell binding
molecule;
placing the disc into an optical reader that supports it on a first side;
rotating the disc
about an axis substantially perpendicular to the first side; scanning the
chamber with
an incident beam of electromagnetic radiation by rotating the disc about an
axis
substantially perpendicular to the first side by moving the incident beam in a
direction
radial to the axis; detecting a return beam of electromagnetic radiation
formed by at
least a part of the incident beam after interacting with the disc; converting
the return
beam into an output signal; analyzing the output signal to determine the
presence of
agglutinated or non-agglutinated cells bound on the capture field; and
determining the
presence of antibodies in the sample.
In certain embodiments of the second aspect, the creating step includes the
creation of two samples, a first sample utilizing Type A1 cells and a second
sample
utilizing Type B cells. In certain embodiments of the second aspect, step (b)
further
comprises the creation of a sample with Type AB cells. In certain embodiments
of the
second aspect, the cell-binding molecule is an anti-human immunoglobulin. In
certain
embodiments of the second aspect, the cell-binding molecule is a lectin or the
cell-
binding molecule is gold.
In another embodiment of the second aspect of the present invention, the
creating step includes mixing Type A and Type B reagent cells together to
produce a
cell mixture. A blood serum or plasma sample is then added to the cell
mixture. The
resulting serum-cell mixture is added to a chamber in an optical bio-disc and
incubated at a pre-determined temperature for a pre-determined time to allow
ample
interaction between the antibodies in the serum and the reagent cells so that
agglutination of any of the typed reagent cells takes place if the appropriate
antibody
is present in the serum sample. The chamber in an optical bio-disc includes
capture
fields which contain antibodies specific for the "A" antigen and one for the
"B' antigen.
These specific antibodies or cell binding proteins may be IgG or IgM. The Type
A
reagent cells will bind to the anti-A antigen capture field and the Type B
reagent cells
will bind to the anti-B antigen capture field. After spinning off the unbound
cells in the
solution the capture fields are analyzed and software may then be used to
determine
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weather the captured cells are agglutinated or single and thus determine the
presence
or absence of antibody to A and/or B antigen.
In certain embodiments of the second aspect, the optical bio-disc is a
reflective
disc or the optical bio-disc is a transmissive disc. In certain embodiments of
the
second aspect, the optical bio-disc comprises a CD, CD-R, DVD-R, or a DVD.
In a third aspect, the invention provides method for determining the presence
of antibodies to an ABO blood group in an individual's blood sample by reverse-
typing
on an optical bio-disc comprising applying a blood sample to at least one
microfluidic
channel or circuit in the optical bio-disc including a separation chamber with
at least
one microfilter, at least one mixing chamber, and at least one capture chamber
or
analysis chamber; spinning the optical bio-disc for a first time at a first
speed to effect
separation of the blood sample into cells and serum in the separation chamber;
spinning the optical bio-disc for a second time at a second speed higher than
the first,
the second speed effecting movement of the serum through the microfluidic
channel
or circuit into a mixing chamber; adding typed reagent cells of a known ABO
blood
group into the mixing chamber containing serum; spinning the optical bio-disc
for a
third time in one direction and alternately in another direction at least once
to effect
mixing of the serum and the cells; incubating the cells in the serum for a
sufficient
period of time to allow antibody-antigen binding; spinning the optical bio-
disc for a
fourth time at a third speed higher than the second, the third speed effecting
movement of the cells into a capture chamber, the capture chamber including
surface
with a molecule that binds cells; incubating the sample in the capture chamber
to
promote cell binding to the chamber surface; spinning the disc for a fifth
time to
remove non-bound cells from the capture field; scanning the chamber with an
incident
beam of electromagnetic radiation by rotating the disc about an axis
substantially
perpendicular to the first side by moving the incident beam in a direction
radial to the
axis; detecting a return beam of electromagnetic radiation formed by at least
a part of
the incident beam after interacting with the disc; converting the return beam
into an
output signal; analyzing the output signal to determine the presence of
agglutinated
cells; and determining the presence of antibodies to a blood group in the
sample.
In certain embodiments of the third aspect, there is a first mixing chamber
connected to a first capture chamber and a second mixing chamber connected to
a
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second capture chamber. In certain embodiments of the third aspect, Type A1
cells
are placed in the first mixing chamber and Type B cells are placed in the
second
mixing chamber. In certain embodiments of this embodiment, the method further
comprising a third mixing chamber connected to a third capture chamber and AB
cells
are added to the third mixing chamber in which AB cells are added.
In another embodiment of the third aspect of the present invention, the Type
A1 and Type B cells are mixed together to produce a cell mixture. The cell
mixture is
then added to the mixing chamber containing the blood serum or plasma and
incubated to allow agglutination of any of the typed reagent cells if their
respective
antibodies are present in the serum sample. The mixing chamber in the optical
bio-
disc is in fluid communication with an analysis chamber which includes capture
fields
that contain antibodies specific for the A antigen and for the B antigen.
These specific
antibodies or cell binding proteins may be IgG or IgM. When the disc is
rotated the
cells in the mixing chamber will move into the anaylsis chamber where the
cells are
captured in their respective capture fields. After spinning off the unbound
cells in the
solution the capture fields are analyzed and software may then be used to
determine
weather the captured cells are agglutinated or single and thus determine the
presence
or absence of antibody to A and/or B antigen.
In certain embodiments of the third aspect, the cell-binding molecule is an
anti-
human immunoglobulin, the cell-binding molecule is a lectin, or the cell-
binding
molecule is gold. In certain embodiments of the third aspect, the optical bio-
disc is a
reflective disc or the optical bio-disc is a transmissive disc. In certain
embodiments of
the third aspect, the optical bio-disc comprises a CD, DVD, CD-R, or DVD-R. In
certain embodiments of the third aspect, the first speed is from about 1 X to
about 3X,
the second speed is greater than 3X but less than about 5X, and the third
speed is
greater than about 5X. (1 X refers to the audio standard for speed).
In a fourth aspect of the current invention, there is provided a method of
performing immunohematology assays including direct and indirect blood typing
and
serum or plasma antibody detection using a bio-matrix. The bio-matrix may be
formed from, but not limited to, cross-linked molecular structures including
polyacrylamide gels, agarose, polydextran, and microspheres such as Dextran
acrylamide spheres, polystyrene microspheres, and glass beads. The cross-
linked
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molecular structures or microspheres may be packed or formed in a micro-
channel in
an optical bio-disc to form the bio-matrix such that the pore size of the bio-
matrix is
sufficiently large to allow passage of single cells and sufficiently small to
a retard and
retain agglutinated cells. The channel may also be filled with an assay
solution
necessary to carry out the desired reaction including buffer, specific
antibodies, and/or
anti human globulin. The creation of the bio-matrix and preparation of the
assay
solution in an optical bio-disc may be prepared and used, for example, as
described in
U.S. Patent No. 5,512,432 to Lapierre, et al, issued on April 30, 1996 and
entitled
"Method Detecting Antigens and/or Antibodies" which is herein incorporated by
reference in its entirety. The fourth aspect of the current invention provides
an
improved platform for the currently used gel test methods, including those
described
in U.S. Patent No. 5,512,432. The gel test method predominantly in current use
was
developed by Lapierre and associates in the early 1930s (Lapierre, Y., et al.,
The Gel
Test: A Neinr V1/ay to Detect Red Cell Antigen-Antibody Reactions, Transfusion
(1990);
30:109).
One distinguishing aspect of the blood typing methods of the present
invention,
is the ability to quantify the degree of agglutination between cells within a
small
chamber. Upon centrifugation, single cells pass through the matrix material
pelleting
at the bottom of the fluidic circuit or analysis chamber whilst agglutinated
cells are
retarded either entirely above the matrix material (strong reaction) or
distributed
through it (weak reaction) as shown below in Figs. 37A, 37B, and 37C.
Quantification of the degree of agglutination is carried out on individual
optical
disc chambers either by visual examination or by a laser scanning mechanism.
This
quantification supports both forward and reverse blood typing assays, and
serum or
plasma antibody detection and testing. The major advantages of implementing
this
method in an optical bio-disc is that it removes the previous need for time
consuming
washing steps and it enables the automation of the analysis of the assay using
an
optical disc reader and its accompanying software.
One advantage of the fourth aspect of the current invention is that by
transposing the matrix based methodology to a multi-chambered optical bio-
disc,
forward and reverse blood typing, serum or plasma antibody detection and
testing,
and other immunohematology assays can be achieved in one convenient multi-
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chambered bio-disc. This in itself will increase the efficiency of screening
multiple
samples and serum antibody panel testing, for example, in the blood banking
and
transfusion fields of use. Moreover by exploiting the imaging capabilities of
the optical
bio-disc drive the degree of agglutination can be determined with a much
greater
degree of accuracy. This capability is of great value in detecting very weak
positive
reactions. Such an optical bio-disc drive is described, for example, in U.S.
Patent
Application Serial No. 10/043,688 entitled "Optical Disc Analysis System
Including
Related Methods for Biological and Medical Imaging" filed January 10, 2002
which is
herein incorporated by reference in its entirety.
In a fifth aspect, the invention provides a method for determining the
presence
of antibodies to a blood group type in an individual by antibody typing on an
optical
bio-disc comprising purifying serum from a blood sample; creating at least one
sample
by mixing serum with cells of a known blood group phenotype or typed reagent
cells;
injecting at least one sample into at least one channel in the optical bio-
disc, thereby
delivering the sample onto a capture field including a incubating the sample
on the
capture field to allow the cells to bind to the cell binding molecule; placing
the disc into
an optical reader that supports it on a first side; rotating the disc about an
axis
substantially perpendicular to the first side; scanning the chamber with an
incident
beam of electromagnetic radiation by rotating the disc about an axis
substantially
perpendicular to the first side by moving the incident beam in a direction
radial to the
axis; detecting a return beam of electromagnetic radiation formed by at least
a part of
the incident beam after interacting with the disc; converting the return beam
into an
output signal; analyzing the output signal to determine the presence of cells
bound to
the capture field; and determining the presence of blood group antibodies.
In certain embodiments of the fifth aspect, the cell-binding molecule is an
anti-
human immunoglobulin. In other embodiments of this aspect, the optical bio-
disc is a
reflective and/or transmissive disc. In certain embodiments of this aspect,
the optical
bio-disc comprises a CD, DVD, CD-R, or DVD-R.
In certain embodiments of the fifth aspect, the cells added are characterized
as
having at least one of the following: an ABO system blood group cell
phenotype, an
Rh system blood group cell phenotype, an MNSs system blood group cell
phenotype,
a P system blood group cell phenotype, a Lutheran system blood group cell
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phenotype, a Kell system blood group cell phenotype, a Lewis system blood
group
cell phenotype, a Duffy system blood group cell phenotype, a Kidd system blood
group cell phenotype, a Fisher system blood group antigen, or a red blood cell
group
antigen from any other group.
In a sixth aspect, the invention provides a method for determining the
presence
of antibodies to a blood group type in an individual's blood sample by
antibody-typing
on an optical bio-disc comprising applying a blood sample to at least one
microfluidic
channel in the optical bio-disc including a separation chamber with at least
one
microfilter, at least one mixing chamber, and at least one capture chamber;
spinning
the optical bio-disc for a first time at a first speed to effect separation of
the blood
sample into cells and serum in the separation chamber; spinning the optical
bio-disc
for a second time at a second speed higher than the first, the second speed
effecting
movement of the serum through the microfluidic channel into a mixing chamber;
adding cells of a known blood group cell phenotype into the mixing chamber
containing serum; spinning the optical bio-disc for a third time in one
direction and
alternately in another direction at least once to effect mixing of the serum
and the
cells; incubating the cells in the serum for a sufficient period of time to
allow antibody-
antigen binding; spinning the optical bio-disc for a fourth time at a third
speed higher
than the second, the third speed effecting movement of the cells into a
capture
chamber, the capture chamber including a surface with an anti-human
immunoglobulin molecule; incubating the sample in the capture chamber to
promote
cell binding to the chamber surface; spinning the optical bio-disc for a fifth
time to
remove non-bound cells; scanning the chamber with an incident beam of
electromagnetic radiation by rotating the disc about an axis substantially
perpendicular to the first side by moving the incident beam in a direction
radial to the
axis; detecting a return beam of electromagnetic radiation formed by at least
a part of
the incident beam after interacting with the disc; converting the return beam
into an
output signal; analyzing the output signal to determine if the cells are
bound; and
determining the presence of blood group antibodies in the sample.
In certain embodiments of this aspect, the cell-binding molecule is a lectin
or
wherein the cell-binding molecule is gold. In other certain embodiments of the
fifth
aspect, the optical bio-disc is a reflective disc or the optical bio-disc is a
transmissive
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disc. In certain embodiments of this embodiment, the optical bio-disc
comprises a
CD, CD-R, DVD, or DVD-R. In certain embodiments of this embodiment, the first
speed is from about 1 X to about 3X, the second speed is greater than 3X but
less
than about 5X, and the third speed is greater than about 5X.
In certain embodiments of the sixth aspect, the cells added are characterized
as having at least one of the following: an ABO system blood group cell
phenotype, an
Rh system blood group cell phenotype, an MNSs system blood group cell
phenotype,
a P system blood group cell phenotype, a Lutheran system blood group cell
phenotype, a Kell system blood group cell phenotype, a Lewis system blood
group
cell phenotype, a Duffy system blood group cell phenotype, a Kidd system blood
group cell phenotype, a Fisher system blood group antigen, or a red blood cell
group
antigen from any other blood group.
In a seventh aspect, the invention provides an apparatus for determining a
blood group type of an individual. The apparatus includes an optical bio-disc
including
at least one capture chamber including a layer including a first capture
antibody, and a
layer including a second capture antibody bound by the first capture antibody,
the
second capture antibody being specific for a blood group antigen; a disc drive
assembly; an optical reader; and software for blood group analysis.
In certain embodiments of the seventh aspect, the capture antibody is an anti-
IgG antibody or the capture antibody is an anti-IgM antibody. In certain
embodiments
of the sixth aspect, the capture antibody is an antibody specific for a red
blood cell
antigen. In certain embodiments of the latter embodiment, red blood cell
antigen is an
ABO system blood group antigen, the red blood cell antigen is an Rh system
blood
group antigen, the red blood cell antigen is an MNSs system blood group
antigen, the
red blood cell antigen is a P system blood group antigen, the red blood cell
antigen is
a Lutheran system blood group antigen, the red blood cell antigen is a Kell
system
blood group antigen, the red blood cell antigen is a Lewis system blood group
antigen
or the red blood cell antigen is a Duffy system blood group antigen, the red
blood cell
antigen is a Kidd system blood group antigen, the red blood cell antigen is a
Fisher
system blood group antigen, or a red blood cell group antigen from any other
group.
In certain embodiments of the sixth aspect, the optical bio-disc is a
reflective disc or
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the optical bio-disc is a transmissive disc or the optical bio-disc comprises
a CD or a
DVD.
In an eighth aspect, the invention provides an optical-bio disc for performing
immunohematology assays. The bio-disc includes a substrate; a separation
chamber
associated with the substrate, the separation chamber including first inlet
port; filter
means associated with the separation chamber; a first mixing chamber in fluid
communication with the separation chamber, the first mixing chamber including
a
second inlet port; a second mixing chamber in fluid communication with the
separation chamber, the second mixing chamber including a third inlet port; a
first
analysis or detection chamber in fluid communication with the first mixing
chamber,
the first analysis or detection chamber including a capture field; and a
second analysis
or detection chamber in fluid communication with the second mixing chamber,
the
second analysis or detection chamber including a capture field. In certain
embodiments of the eighth aspect, the optical bio-disc does not contain a
second inlet
port leading to the mixing chamber (e.g., if the material that would otherwise
be
delivered via the second inlet port is supplied ahead of time in the mixing
chamber, in
freeze-dried or other form). ,
In certain embodiments of the eighth aspect, when a sample of blood is
directed into the separation chamber through the inlet port and the disc is
rotated at a
first speed, the filter means separates white blood cells, red blood cells,
and platelets
from the blood sample to provide a sample of serum. In a further embodiment,
when
the disc is rotated at a second speed, the sample of serum is directed into
the first
and second mixing chambers. In another embodiment, the inlet port of the first
mixing
chamber is employed to direct cells of a first type into the first mixing
chamber, and
the inlet port of the second mixing chamber is employed to direct cells of a
second
type into the second mixing chamber. In other certain embodiments, when the
disc is
rotated at a third speed, a mixture of serum and cells of the first type is
directed into
the first analysis or detection chamber, and a mixture of serum and cells of
the
second type is directed into the second analysis or detection chamber.
Certain embodiments of the eighth aspect provide for disc rotation in a
predetermined manner to mix the cells of the first type with serum in the
first mixing
chamber, and mix the cells of the second type with serum in the second mixing
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chamber. In certain embodiments, the predetermined manner of rotating the disc
includes alternately rotating the disc in one direction and then an opposite
direction to
thereby create an agitation action to promote mixing of serum and cells.
In certain embodiments of the eighth aspect, the capture field in the first
analysis or detection chamber includes a first type of capture agent
implemented to
capture specific cells having any affinity therefor. In other certain
embodiments, the
capture field in the second analysis or detection chamber includes a second
type of
capture agent implemented to capture specific cells having any affinity
therefor.
In certain embodiments of the eighth aspect, an incident beam of radiant
energy is directed into the first analysis or detection chamber to determine
whether
any cells were captured by the first type of capture agent. In other
embodiments, an
incident beam of radiant energy is directed into the second analysis or
detection
chamber to determine whether any cells were captured by the second type of
capture
agent.
In certain embodiments of the eighth aspect, the first type of capture agent
is
an anti-human immunoglobulin having an affinity for an antibody bound to the
cells or
a non-cell specific molecule that binds a molecule on the surface of all red
blood cells.
In certain embodiments of the eighth aspect, the second type of capture agent
is an
anti-human immunoglobulin having an affinity for an antibody bound to the
cells or a
non-cell specific molecule that binds a molecule on the surface of all red
blood cells.
In certain embodiments of the eighth aspect, the separation chamber, the first
and second mixing chambers, and the first and second analysis or detection
chambers are formed in the substrate. In other certain embodiments of the
seventh
aspect, the separation chamber, the first and second mixing chambers, and the
first
and second analysis or detection chambers are formed in a cap bonded to the
substrate. In yet other certain embodiments of the eighth aspect, the
separation
chamber, the first and second mixing chambers, and the first and second
analysis or
detection chambers are formed in a channel layer bonded between a cap portion
and
the substrate. In certain embodiments of the seventh aspect, the separation
chamber, the first and second mixing chambers, and the first and second
analysis or
detection chambers are partially formed in a cap portion and partially formed
in the
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substrate, the cap portion and the substrate being bonded together in register
to
thereby fully form the chambers.
In certain embodiments of the eighth aspect, the optical bio-disc further
includes information encoded in an information layer readable by a disc drive.
In
certain embodiments thereof, the encoded information is used to rotate the
disc in a
prescribed manner. In certain embodiments of the eighth aspect, the
information
layer is reflective. In yet other embodiments of this aspect, the information
layer is
semi-reflective.
In a ninth aspect, the invention provides an optical-bio disc for performing
immunohematology assays. The bio-disc includes a substrate; a separation
chamber
associated with the substrate, the separation chamber including first inlet
port; filter
means associated with the separation chamber; a first mixing chamber in fluid
communication with the separation chamber, the first mixing chamber including
a
second inlet port; a second mixing chamber in fluid communication with the
separation chamber, the second mixing chamber including a third inlet port; a
first
analysis or detection chamber in fluid communication with the first mixing
chamber,
the first analysis or detection chamber including a bio-matrix; and a second
analysis
or detection chamber in fluid communication with the second mixing chamber,
the
second analysis or detection chamber including a bio-matrix. In certain
embodiments
of the ninth aspect, the optical bio-disc does not contain a second inlet port
leading to
the mixing chamber (e.g., if the material that would otherwise be delivered
via the
second inlet port is supplied ahead of time in the mixing chamber, in freeze-
dried or
other form).
In certain embodiments of the ninth aspect, when a sample of blood is directed
into the separation chamber through the inlet port and the disc is rotated at
a first
speed, the filter means separates white blood cells, red blood cells, and
platelets from
the blood sample to provide a sample of serum. In a further embodiment, when
the
disc is rotated at a second speed, the sample of serum is directed into the
first and
second mixing chambers. In another embodiment, the inlet port of the first
mixing
chamber is employed to direct cells of a first type into the first mixing
chamber, and
the inlet port of the second mixing chamber is employed to direct cells of a
second
type into the second mixing chamber. In other certain embodiments, when the
disc is
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rotated at a third speed, a mixture of serum and cells of the first type is
directed into
the first analysis or detection chamber, and a mixture of serum and cells of
the
second type is directed into the second analysis or detection chamber.
Certain embodiments of the ninth aspect provide for disc rotation in a
predetermined manner to mix the cells of the first type with serum in the
first mixing
chamber, and mix the cells of the second type with serum in the second mixing
chamber. In certain embodiments, the predetermined manner of rotating the disc
includes alternately rotating the disc in one direction and then an opposite
direction to
thereby create an agitation action to promote mixing of serum and cells.
In certain embodiments of the ninth aspect, an incident beam of radiant energy
is directed into the first analysis or detection chamber to determine the
location of
cells and amount of agglutination within the first analysis or detection
chamber. In
other embodiments, an incident beam of radiant energy is directed into the
second
analysis or detection chamber to determine the location of cells and amount of
agglutination within the second analysis or detection chamber.
In certain embodiments of the ninth aspect, the first and second analysis or
detection chambers may include anti-human immunoglobulin having an affinity
for an
antibody bound to the cells or a non-cell specific molecule that binds a
molecule on
the surface of all red blood cells.
In certain embodiments of the ninth aspect, the separation chamber, the first
and second mixing chambers, and the first and second analysis or detection
chambers are formed in the substrate. In other certain embodiments of the
seventh
aspect, the separation chamber, the first and second mixing chambers, and the
first
and second analysis or detection chambers are formed in a cap bonded to the
substrate. In yet other certain embodiments of the ninth aspect, the
separation
chamber, the first and second mixing chambers, and the first and second
analysis or
detection chambers are formed in a channel layer bonded between a cap portion
and
the substrate. In certain embodiments of the ninth aspect, the separation
chamber,
the first and second mixing chambers, and the first and second analysis or
detection
chambers are partially formed in a cap portion and partially formed in the
substrate,
the cap portion and the substrate being bonded together in register to thereby
fully
form the chambers.
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In certain embodiments of the ninth aspect, the optical bio-disc further
includes
information encoded in an information layer readable by a disc drive. In
certain
embodiments thereof, the encoded information is used to rotate the disc in a
prescribed manner. In certain embodiments of the ninth aspect, the information
layer
is reflective. In yet other embodiments of this aspect, the information layer
is semi-
reflective.
In an tenth aspect, the invention provides a method for manufacturing a disc
comprising: providing over a substrate of the disc an encoded informational
layer;
forming target areas; providing a capture layer in the target areas; attaching
at least
one capture agent. In certain embodiments, the encoded informational layer is
a
reflective layer, and the target areas are regions etched from the reflective
layer. In
certain embodiments, the encoded informational layer is a partially reflective
and
partially transmissive layer, and the target areas are regions adjacent to the
informational layer.
In an eleventh aspect, the invention provides a method for manufacturing a
disc comprising: providing over a substrate of the disc an encoded
informational layer;
providing a cover disc; forming fluidic circuits between the cover disc and
the
substrate; and forming a bio-matrix with a pre-determined pore size within
said fluidic
circuits. In certain embodiments, the encoded informational layer is a
reflective layer,
and the target areas are regions etched from the reflective layer. In certain
embodiments, the encoded informational layer is a partially reflective and
partially
transmissive layer, and the target areas are regions adjacent to the
informational
layer.
The above described and many other features and attendant advantages of the
present invention will become better understood by reference to the following
detailed
description when taken in conjunction with the accompanying drawing figures
and
experimental examples.
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BRIEF DESCRIPTION OF THE DRAWING FIGURES
Further objects of the present invention together with additional features
contributing thereto and advantages accruing therefrom will be apparent from
the
following description of the preferred embodiments of the invention which are
shown
in the accompanying drawing with like reference numerals indicating like
components
throughout, wherein:
Fig. 1 is a pictorial representation of a bio-disc system according to the
present
invention;
Fig. 2 is an exploded perspective view of a reflective bio-disc as utilized in
conjunction with the present invention;
Fig. 3 is a top plan view of the disc shown in Fig. 2;
Fig. 4 is a perspective view of the disc illustrated in Fig. 2 with cut-away
sections showing the different layers of the disc;
Fig. 5 is an exploded perspective view of one embodiment of a transmissive
bio-disc as employed in conjunction with the present invention;
Fig. 6 is a perspective view representing the disc shown in Fig. 5 with a cut-
away section illustrating the functional aspects of a semi-reflective layer of
the disc;
Fig. 7 is a graphical representation showing the relationship between
thickness
and transmission of a thin gold film;
Fig. 8 is a top plan view of the disc shown in Fig. 5;
Fig. 9 is a perspective view of the disc illustrated in Fig. 5 with cut-away
sections showing the different layers of the disc including the type of semi-
reflective
layer shown in Fig. 6;
Fig. 10 is a perspective and block diagram representation illustrating the
system of Fig. 1 in more detail;
Fig. 11 is a partial cross sectional view taken perpendicular to a radius of
the
reflective optical bio-disc illustrated in Figs. 2, 3, and 4 showing a flow
channel formed
therein;
Fig. 12 is a partial cross sectional view taken perpendicular to a radius of
the
transmissive optical bio-disc illustrated in Figs. 5, 8, and 9 showing a flow
channel
formed therein and a top detector;
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Fig. 13 is a partial longitudinal cross sectional view of the reflective
optical bio-
disc shown in Figs. 2, 3, and 4 illustrating a wobble groove formed therein;
Fig. 14 is a partial longitudinal cross sectional view of the transmissive
optical
bio-disc illustrated in Figs. 5, 8, and 9 showing a wobble groove formed
therein and a
top detector;
Fig. 15 is a view similar to Fig. 11 showing the entire thickness of the
reflective
disc and the initial refractive property thereof;
Fig. 16 is a view similar to Fig. 12 showing the entire thickness of the
transmissive disc and the initial refractive property thereof;
Fig. 17 is a pictorial flow diagram presenting one example of a forward ABO/Rh
blood typing method of the invention;
Fig. 18 is a pictorial presenting a general schematic of the cell capture
technologies of the invention;
Fig. 19 is a pictorial schematic presenting the biotin/streptavidin-based cell
capture technologies of the invention;
Fig. 20 is a plan view illustrating data output in the form of a bar code;
Figs. 21 A to 21 F is a schematic presenting a series of cross sections
demonstrating the preparation of one example of a bio-disc of the invention;
Fig. 22 is a pictorial flow diagram illustrating different methods of reverse
typing
for the ABO/Rh blood groups with sample preparation off-disc and sample
analysis on
disc;
Fig. 23 is another pictorial flov~ diagram illustrating different methods of
reverse
typing for the ABO/Rh blood groups with sample preparation off-disc and sample
analysis on disc;
Figs. 24A-24C are cross-sectional side views of an optical bio-disc showing
the
cell binding during the reverse typing test when antibodies to an ABO/Rh blood
group
antigen are present;
Figs. 25A to 25C are cross-sectional side views of an optical bio-disc showing
cell binding during the reverse typing test when no antibodies to an ABO/Rh
blood
group antigen are present;
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Figs. 26A to 26C are cross sectional side views of an optical bio-disc showing
differential cell binding during the reverse typing test where Type A and Type
B cells
are mixed together and specific anti-A and anti-B capture fields are present;
Fig. 27 is a pictorial flow diagram presenting the method of reverse ABO/Rh
typing wherein sample preparation and processing are all done on the optical
bio-disc;
Fig. 28 is a pictorial flow diagram demonstrating the method of antibody
typing
for blood groups other than the ABO/Rh types with sample preparation off-disc
and
sample analysis on disc;
Figs. 29A-29C are cross-sectional side views of an optical bio-disc including
red blood cells bound by antibodies coming into contact with, and being
captured by
the capture field;
Figs. 30A-30C are cross-sectional side views of an optical bio-disc including
red blood cells not bound by antibodies coming into contact with, and not
being
captured by the capture field;
Fig. 31 is a pictorial flow diagram presenting the method of antibody typing
wherein sample preparation and processing are all done on the optical bio-
disc;
Fig. 32 is an enlarged plan view representing one example of a microfluidic
channel containing inlet ports, a separation chamber, mixing chambers, capture
or
analysis chambers, and vent ports;
Fig. 33 is a pictorial of a computer monitor screen shot presenting an output
of
an ABO blood typing test;
Fig. 34 illustrates a flow diagram of a procedure used in blood typing
analysis;
Fig. 35 illustrates a bio-matrix packed in a microfluidic channel or circuit
containing an assay solution;
Figs. 36A and 36B depict the addition of particles or cells and formation of
agglutinates in a microfluidic channel containing a pre-formed bio-matrix;
Figs. 37A, 37B, and 37C show three different patterns of particle separation
using a bio-matrix respectively depicting strong, weak, and negative
reactions;
Fig. 38 is a top plan view of another embodiment of a transmissive optical bio-
disc showing semi-circular, equi-radial fluidic circuits;
Fig. 39 is an enlarged detailed view of a portion of the equi-radial fluidic
circuit
of the disc shown in Fig. 38; and
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Fig. 40 is an enlarged detailed view of a portion of yet another embodiment of
the transmissive disc with proximal and distal equi-radial fluidic circuits.
DETAILED DESCRIPTION OF THE INVENTION
The patents and publications cited herein reflect the level of knowledge in
the
art and are hereby incorporated by reference in their entirety. Any conflict
between
any teaching of such references and this specification shall be resolved in
favor of the
latter.
The invention described herein provides diagnostic assays based on cell-
capture and/or cell separation technologies adapted to an optical bio-disc and
methods and compositions related thereto.
Drive System and Related Discs
Fig. 1 is a perspective view of an optical bio-disc 110 according to the
present
invention as implemented to conduct the cell counts and differential cell
counts
disclosed herein. The present optical bio-disc 110 is shown in conjunction
with an
optical disc drive 112 and a display monitor 114. Further details relating to
this type of
disc drive and disc analysis system are disclosed in commonly assigned and
copending U.S. Patent Application Serial No. 10/008,156 entitled "Disc Drive
System
and Methods for Use with Bio-discs" filed November 9, 2001 and U.S. Patent
Application Serial No. 101043,688 entitled "Optical Disc Analysis System
Including
Related Methods For Biological and Medical Imaging" filed January 10, 2002,
both of
which are herein incorporated by reference.
Fig. 2 is an exploded perspective view of the principal structural elements of
one embodiment of the optical bio-disc 110. Fig. 2 is an example of a
reflective zone
optical bio-disc 110 (hereinafter "reflective disc") that may be used in the
present
invention. The principal structural elements include a cap portion 116, an
adhesive
member or channel layer 118, and a substrate 120. The cap portion 116 includes
one
or more inlet ports 122 and one or more vent ports 124. The cap portion 116
may be
formed from polycarbonate and is preferably coated with a reflective surface
146 (Fig.
4) on the bottom thereof as viewed from the perspective of Fig. 2. In the
preferred
embodiment, trigger marks or markings 126 are included on the surface of the
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reflective layer 142 (Fig. 4). Trigger markings 126 may include a clear window
in all
three layers of the bio-disc, an opaque area, or a reflective or semi-
reflective area
encoded with information that sends data to a processor 166, as shown Fig. 10,
that
in turn interacts with the operative functions of the interrogation or
incident beam 152,
Figs. 6 and 10.
The second element shown in Fig. 2 is an adhesive member or channel layer
118 having fluidic circuits 128 or U-channels formed therein. The fluidic
circuits 128
are formed by stamping or cutting the membrane to remove plastic film and form
the
shapes as indicated. Each of the fluidic circuits 128 includes a flow channel
130 and
a return channel 132. Some of the fluidic circuits 128 illustrated in Fig. 2
include a
mixing chamber 134. Two different types of mixing chambers 134 are
illustrated. The
first is a symmetric mixing chamber 136 that is symmetrically formed relative
to the
flow channel 130. The second is an off-set mixing chamber 138. The off-set
mixing
chamber 138 is formed to one side of the flow channel 130 as indicated.
The third element illustrated in Fig. 2 is a substrate 120 including target or
capture fields 140. The substrate 120 is preferably made of polycarbonate and
has a
reflective layer 142 deposited on the top thereof, Fig. 4. The target zones
140 are
formed by removing the reflective layer 142 in the indicated shape or
alternatively in
any desired shape. Alternatively, the target zone 140 may be formed by a
masking
technique that includes masking the target zone 140 area before applying the
reflective layer 142. The reflective layer 142 may be formed from a metal such
as
aluminum or gold.
Fig. 3 is a top plan view of the optical bio-disc 110 illustrated in Fig. 2
with the
reflective layer 142 on the cap portion 116 shown as transparent to reveal the
fluidic
circuits, the target zones 140, and trigger markings 126 situated within the
disc.
Fig. 4 is an enlarged perspective view of the reflective zone type optical bio-
disc 110 according to one embodiment of the present invention. This view
includes a
portion of the various layers thereof, cut away to illustrate a partial
sectional view of
each principal layer, substrate, coating, or membrane. Fig. 4 shows the
substrate 120
that is coated with the reflective layer 142. An active layer 144 is applied
over the
reflective layer 142. In the preferred embodiment, the active layer 144 may be
formed
from polystyrene. Alternatively, polycarbonate, gold, activated glass,
modified glass,
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or modified polystyrene, for example, polystyrene-co-malefic anhydride, may be
used.
In addition, hydrogels can be used. Alternatively as illustrated in this
embodiment,
the plastic adhesive member 118 is applied over the active layer 144. The
exposed
section of the plastic adhesive member 118 illustrates the cut out or stamped
U-
shaped form that creates the fluidic circuits 128. The final principal
structural layer in
this reflective zone embodiment of the present bio-disc is the cap portion
116. The
cap portion 116 includes the reflective surface 146 on the bottom thereof. The
reflective surface 146 may be made from a metal such as aluminum or gold.
Referring now to Fig. 5, there is shown an exploded perspective view of the
principal structural elements of a transmissive type of optical bio-disc 110
according to
the present invention. The principal structural elements of the transmissive
type of
optical bio-disc 110 similarly include the cap portion 116, the adhesive or
channel
member 118, and the substrate 120 layer. The cap portion 116 includes one or
more
inlet ports 122 and one or more vent ports 124. The cap portion 116 may be
formed
from a polycarbonate layer. Optional trigger markings 126 may be included on
the
surface of a thin semi-reflective layer 143, as best illustrated in Figs. 6
and 9. Trigger
markings 126 may include a clear window in all three layers of the bio-disc,
an opaque
area, or a reflective or semi-reflective area encoded with information that
sends data
to the processor 166, Fig. 10, which in turn interacts with the operative
functions of the
interrogation beam 152, Figs. 6 and 10.
The second element shown in Fig. 5 is the adhesive member or channel layer
118 having fluidic circuits 128 or U-channels formed therein. The fluidic
circuits 128
are formed by stamping or cutting the membrane to remove plastic film and form
the
shapes as indicated. Each of the fluidic circuits 128 includes the flow
channel 130
and the return channel 132. Some of the fluidic circuits 128 illustrated in
Fig. 5
include the mixing chamber 134. Two different types of mixing chambers 134 are
illustrated. The first is the symmetric mixing chamber 136 that is
symmetrically
formed relative to the flow channel 130. The second is the off-set mixing
chamber
138. The off-set mixing chamber 138 is formed to one side of the flow channel
130 as
indicated.
The third element illustrated in Fig. 5 is the substrate 120 which may include
the target or capture fields 140. The substrate 120 is preferably made of
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polycarbonate and has the thin semi-reflective layer 143 deposited on the top
thereof,
Fig. 6. The semi-reflective layer 143 associated with the substrate 120 of the
disc 110
illustrated in Figs. 5 and 6 is significantly thinner than the reflective
layer 142 on the
substrate 120 of the reflective disc 110 illustrated in Figs. 2, 3 and 4. The
thinner
semi-reflective layer 143 allows for some transmission of the interrogation
beam 152
through the structural layers of the transmissive disc as shown in Figs. 6 and
12. The
thin semi-reflective layer 143 may be formed from a metal such as aluminum or
gold.
Fig. 6 is an enlarged perspective view of the substrate 120 and semi-
reflective
layer 143 of the transmissive embodiment of the optical bio-disc 110
illustrated in Fig.
5. The thin semi-reflective layer 143 may be made from a metal such as
aluminum or
gold. In the preferred embodiment, the thin semi-reflective layer 143 of the
transmissive disc illustrated in Figs. 5 and 6 is approximately 10-300 A thick
and does
not exceed 400 A. This thinner semi-reflective layer 143 allows a portion of
the
incident or interrogation beam 152 to penetrate and pass through the semi-
reflective
layer 143 to be detected by a top detector 158, Figs. 10 and 12, while some of
the
light is reflected or returned back along the incident path. As indicated
below, Table 1
presents the reflective and transmissive characteristics of a gold film
relative to the
thickness of the film. The gold film layer is fully reflective at a thickness
greater than
800 A. While the threshold density for transmission of light through the gold
film is
approximately 400 A.
In addition to Table 1, Fig. 7 provides a graphical representation of the
inverse
relationship of the reflective and transmissive nature of the thin semi-
reflective layer
143 based upon the thickness of the gold. Reflective and transmissive values
used in
the graph illustrated in Fig. 7 are absolute values.
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TABLE 1
Au film Reflection and Transmission (Absolute Valued
Thickness ThicknessReflectance Transmittance
An stroms nm
0 0 0.0505 0.9495
50 5 0.1683 0.7709
100 10 0.3981 0.5169
150 15 0.5873 0.3264
200 20 0.7142 0.2057
250 25 0.7959 0.1314
300 30 0.8488 0.0851
350 35 0.8836 0.0557
400 40 0.9067 0.0368
450 45 0.9222 0.0244
500 50 0.9328 0.0163
550 55 0.9399 0.0109
600 60 0.9448 0.0073
650 65 0.9482 0.0049
700 70 0.9505 0.0033
750 75 0.9520 0.0022
800 80 0.9531 0.0015
With reference next to Fig. 8, there is shown a top plan view of the
transmissive
type optical bio-disc 110 illustrated in Figs. 5 and 6 with the transparent
cap portion
116 revealing the fluidic channels, the trigger markings 126, and the target
zones 140
as situated within the disc.
Fig. 9 is an enlarged perspective view of the optical bio-disc 110 according
to
the transmissive disc embodiment of the present invention. The disc 110 is
illustrated
with a portion of the various layers thereof cut away to show a partial
sectional view of
each principal layer, substrate, coating, or membrane. Fig. 9 illustrates a
transmissive
disc format with the clear cap portion 116, the thin semi-reflective layer 143
on the
substrate 120, and trigger markings 126. In this embodiment, trigger markings
126
include opaque material placed on the top portion of the cap. Alternatively
the trigger
marking 126 may be formed by clear, non-reflective windows etched on the thin
reflective layer 143 of the disc, or any mark that absorbs or does not reflect
the signal
coming from the trigger detector 160, Fig. 10. Fig. 9 also shows, the target
zones 140
formed by marking the designated area in the indicated shape or alternatively
in any
desired shape. Markings to indicate target zone 140 may be made on the thin
semi-
reflective layer 143 on the substrate 120 or on the bottom portion of the
substrate 120
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(under the disc). Alternatively, the target zones 140 may be formed by a
masking
technique that includes masking the entire thin semi-reflective layer 143
except the
target zones 140. In this embodiment, target zones 140 may be created by silk
screening ink onto the thin semi-reflective layer 143. In the transmissive
disc format
illustrated in Figs. 5, 8, and 9, the target zones 140 may alternatively be
defined by
address information encoded on the disc. In this embodiment, target zones 140
do
. not include a physically discernable edge boundary. ,
The substrate layer may be impressed with a spiral track that starts at an
innermost readable portion of the disc and then spirals out to an outermost
readable
portion of the disc. In a non-recordable CD, this track is made up of a series
of
embossed pits with varying length, each typically having a depth of
approximately
one-quarter the wavelength of the light that is used to read the disc. The
varying
lengths and spacing between the pits encode the operational data. The spiral
groove
of a recordable CD-like disc has a detectable dye rather than pits. This is
where the
operation information, such as the rotation rate, is recorded. Depending on
the test,
assay, or investigational protocol, the rotation rate may be variable with
intervening or
consecutive periods of acceleration, constant speed, and deceleration. These
periods
may be closely controlled both as to speed and time of rotation to provide,
for
example, mixing, agitation, or separation of fluids and suspensions with
agents,
reagents, antibodies, or other materials.
With continuing reference to Fig. 9, an active layer 144 is illustrated as
applied
over the thin semi-reflective layer 143. In the preferred embodiment, the
active layer
144 is a 40 to 200 pm thick layer of 2% polystyrene. Alternatively,
polycarbonate,
gold, activated glass, modified glass, or modified polystyrene, for example,
polystyrene-co-malefic anhydride, may be used. In addition, hydrogels can be
used.
As illustrated in this embodiment, the plastic adhesive member 118 is applied
over the
active layer 144. The exposed section of the plastic adhesive member 118
illustrates
the cut out or stamped U-shaped form that creates the fluidic circuits 128.
The final principal structural layer in this transmissive embodiment of the
present bio-disc 110 is the clear, non-reflective cap portion 116 that
includes inlet
ports 122 and vent ports 124.
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Referring next to Fig. 10, there is shown an optical disc reader system. This
system may be a conventional reader for CD, CD-R, DVD, DVD-R or other known
comparable optical disc format, a modified version of such a drive, or a
completely
distinct dedicated device. The basic components are a motor for rotating the
disc, a
light system for providing light, and a detection system for detecting light.
Fig. 10 is a perspective block diagram of an optical disc reader illustrating
optical components 148, a light source 150 that produces the incident or
interrogation
beam 152, a return beam 154, and a transmitted beam 156. In the case of the
reflective bio-disc illustrated in Fig. 4, the return beam 154 is reflected
from the
reflective surface 146 of the cap portion 116 of the optical bio-disc 110. In
this
reflective embodiment of the present optical bio-disc 110, the return beam 154
is
detected and analyzed for the presence of signal elements by a bottom detector
157.
In the transmissive bio-disc format, on the other hand, the transmitted beam
156 is
detected, by a top detector 158, and is also analyzed for the presence of
signal
elements. In the transmissive embodiment, a photo detector may be used as a
top
detector 158.
Fig. 10 also shows a hardware trigger mechanism that includes the trigger
markings 126 on the disc and a trigger detector 160. The hardware triggering
mechanism is used in both reflective bio-discs (Fig. 4) and transmissive bio-
discs (Fig.
9). The triggering mechanism allows the processor 166 to collect data only
when the
interrogation beam 152 is on a respective target zone 140. Furthermore, in the
transmissive bio-disc system, a software trigger may also be used. The
software
trigger uses the bottom detector to signal the processor 166 to collect data
as soon as
the interrogation beam 152 hits the edge of a respective target zone 140. Fig.
10
further illustrates a drive motor 162 and a controller 164 for controlling the
rotation of
the optical bio-disc 110. Fig. 10 also shows the processor 166 and analyzer
168
implemented in the alternative for processing the return beam 154 and
transmitted
beam 156 associated the transmissive optical bio-disc.
Numerous designs and configurations of an optical pickup and associated
electronics may be used in the context of the embodiments of the present
invention.
Further details and alternative designs for compact discs and readers are
described in
Compact Disc Technology, by Nakajima and Ogawa, IOS Press, Inc. (1992); The
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Compact Disc Handbook, Digital Audio and Compact Disc Technology, by Baert et
al.
(eds.), Books Britain (1995); and CD-ROM Professional's CD-Recordable
Handbook:
The Complete Guide to Practical Desktop CD, Starrett et al. (eds.),
ISBN:0910965188
(1996); all of which are incorporated herein in their entirety by reference.
The disc drive assembly is thus employed to rotate the disc, read and process
any encoded operational information stored on the disc, analyze the liquid,
chemical,
biological, or biochemical investigational features in an assay region of the
disc, to
write information to the disc either before or after the material in the assay
zone is
analyzed by the read beam of the drive or deliver the information via various
possible
interfaces, such as Ethernet to a user, database, or anywhere the information
could
be utilized.
As shown in Fig. 11, there is presented a partial cross sectional view of the
reflective disc embodiment of the optical bio-disc 110 according to the
present
invention. Fig. 11 illustrates the substrate 120 and the reflective layer 142.
As
indicated above, the reflective layer 142 may be made from a material such as
aluminum, gold or other suitable reflective material. In this embodiment, the
top
surface of the substrate 120 is smooth. Fig. 11 also shows the active layer
144
applied over the reflective layer 142. As also shown in Fig. 11, the target
zone 140 is
formed by removing an area or portion of the reflective layer 142 at a desired
location
or, alternatively, by masking the desired area prior to applying the
reflective layer 142.
As further illustrated in Fig. 11, the plastic adhesive member 118 is applied
over the
active layer 144. Fig. 11 also shows the cap portion 116 and the reflective
surface
146 associated therewith. Thus when the cap portion 116 is applied to the
plastic
adhesive member 118 including the desired cutout shapes, flow channel 130 is
thereby formed. As indicated by the arrowheads shown in Fig. 11, the path of
the
incident beam 152 is initially directed toward the substrate 120 from below
the disc
110. The incident beam then focuses at a point proximate the reflective layer
142.
Since this focusing takes place in the target zone 140 where a portion of the
reflective
layer 142 is absent, the incident continues along a path through the active
layer 144
and into the flow channel 130. The incident beam 152 then continues upwardly
traversing through the flow channel to' eventually fall incident onto the
reflective
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surface 146. At this point, the incident beam 152 is returned or reflected
back along
the incident path and thereby forms the return beam 154.
Fig. 12 is a partial cross sectional view of the transmissive embodiment of
the
bio-disc 110 according to the present invention. Fig. 12 illustrates a
transmissive disc
format with the clear cap portion 116 and the thin semi-reflective layer 143
on the
substrate 120. Fig. 12 also shows the active layer 144 applied over the thin
semi-
reflective layer 143. In the preferred embodiment, the transmissive disc has
the thin
semi-reflective layer 143 made from a metal such as aluminum or gold
approximately
100-300 Angstroms thick and does not exceed 400 Angstroms. This thin semi-
reflective layer 143 allows a portion of the incident or interrogation beam
152, from the
light source 150, Fig. 10, to penetrate and pass upwardly through the disc to
be
detected by a top detector 158, while some of the light is reflected back
along the
same path as the incident beam but in the opposite direction. In this
arrangement, the
return or reflected beam 154 is reflected from the semi-reflective layer 143.
Thus in
this manner, the return beam 154 does not enter into the flow channel 130. The
reflected light or return beam 154 may be used for tracking the incident beam
152 on
pre-recorded information tracks formed in or on the semi-reflective layer 143
as
described in more detail in conjunction with Figs. 13 and 14. In the disc
embodiment
illustrated in Fig. 12, a physically defined target zone 140 may or may not be
present.
Target zone 140 may be created by direct markings made on the thin semi-
reflective
layer 143 on the substrate 120. These marking may be formed using silk
screening or
any equivalent method. In the alternative embodiment where no physical indicia
are
employed to define a target zone (such as, for example, when encoded software
addressing is utilized) the flow channel 130 in effect may be employed as a
confined
target area in which inspection of an investigational feature is conducted.
Fig. 13 is a cross sectional view taken across the tracks of the reflective
disc
embodiment of the bio-disc 110 according to the present invention. This view
is taken
longitudinally along a radius and flow channel of the disc. Fig. 13 includes
the
substrate 120 and the reflective layer 142. In this embodiment, the substrate
120
includes a series of grooves 170. The grooves 170 are in the form of a spiral
extending from near the center of the disc toward the outer edge. The grooves
170
are implemented so that the interrogation beam 152 may track along the spiral
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grooves 170 on the disc. This type of groove 170 is known as a "wobble
groove". A
bottom portion having undulating or wavy sidewalls forms the groove 170, while
a
raised or elevated portion separates adjacent grooves 170 in the spiral. The
reflective
layer 142 applied over the grooves 170 in this embodiment is, as illustrated,
conformal
in nature. Fig. 13 also shows the active layer 144 applied over the reflective
layer
142. As shown in Fig. 13, the target zone 140 is formed by removing an area or
portion of the reflective layer 142 at a desired location or, alternatively,
by masking the
desired area prior to applying the reflective layer 142. As further
illustrated in Fig. 13,
the plastic adhesive member 118 is applied over the active layer 144. Fig. 13
also
shows the cap portion 116 and the reflective surface 146 associated therewith.
Thus,
when the cap portion 116 is applied to the plastic adhesive member 118
including the
desired cutout shapes, the flow channel 130 is thereby formed.
Fig. 14 is a cross sectional view taken across the tracks of the transmissive
disc embodiment of the bio-disc 110 according to the present invention as
described
in Fig. 12, for example. This view is taken longitudinally along a radius and
flow
channel of the disc. Fig. 14 illustrates the substrate 120 and the thin semi-
reflective
layer 143. This thin semi-reflective layer 143 allows the incident or
interrogation beam
152, from the light source 150, to penetrate and pass through the disc to be
detected
by the top detector 158, while some of the light is reflected back in the form
of the
return beam 154. The thickness of the thin semi-reflective layer 143 is
determined by
the minimum amount of reflected light required by the disc reader to maintain
its
tracking ability. The substrate 120 in this embodiment, like that discussed in
Fig. 13,
includes the series of grooves 170. The grooves 170 in this embodiment are
also
preferably in the form of a spiral extending from near the center of the disc
toward the
outer edge. The grooves 170 are implemented so that the interrogation beam 152
may track along the spiral. Fig. 14 also shows the active layer 144 applied
over the
thin semi-reflective layer 143. As further illustrated in Fig. 14, the plastic
adhesive
member or channel layer 118 is applied over the active layer 144. Fig. 14 also
shows
the cap portion 116 without a reflective surface 146. Thus, when the cap is
applied to
the plastic adhesive member 118 including the desired cutout shapes, the flow
channel 130 is thereby formed and a part of the incident beam 152 is allowed
to pass
therethrough substantially unreflected.
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Fig. 15 is a view similar to Fig. 11 showing the entire thickness of the
reflective disc
and the initial refractive property thereof. Fig. 16 is a view similar to Fig.
12 showing
the entire thickness of the transmissive disc and the initial refractive
property thereof.
Grooves 170 are not seen in Figs. 15 and 16 since the sections are cut along
the
grooves 170. Figs. 15 and 16 show the presence of the narrow flow channel 130
that
is situated perpendicular to the grooves 170 in these embodiments. Figs. 13,
14, 15,
and 16 show the entire thickness of the respective reflective and transmissive
discs.
In these figures, the incident beam 152 is illustrated initially interacting
with the
substrate 120 which has refractive properties that change the path of the
incident
beam as illustrated to provide focusing of the beam 152 on the reflective
layer 142 or
the thin semi-reflective layer 143.
The invention is described below as it relates to clinical diagnostic assays
based on cell-capture and/or cell separation technologies as employed on an
optical
bio-disc described herein. Various embodiments of this aspect of the invention
are
directed to blood-typing and antibody typing diagnostic assays.
Blood Typina Assays
Referring to Fig. 17, there is illustrated a system for blood typing or the
detection of antibodies directed against a particular blood type. The system
includes
methods for the collection and processing of blood for diagnostic purposes. In
the
embodiment presented, whole blood may be collected by standard finger stick.
Ten
microliters of this sample is then diluted with 80 microliters of phosphate
buffered
saline (PBS) and 10 microliters of an anticoagulent (e.g., heparin or ACD).
The
diluted sample is then introduced through the inlet port 122 into the optical
bio-disc
110. The optical bio-disc can be a reflective disc (Fig. 4) or transmissive
disc (Fig. 9).
The optical bio-disc has at least one chamber with a target zone 140 and
capture
fields contained therein. After a sufficient incubation period, e.g. 5
minutes, the optical
bio-disc is loaded onto ~ an optical disc reader 112 and the disc is spun for
approximately 5 minutes. At the end of the spin, the disc is then analyzed and
the
information is collected and transferred to a system for the data processing.
After
processing the collected data, results of the diagnostic assay are transferred
to a
monitor 114 to display output results. Additional details are provided in
Example 2.
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An advantage of the system of the present invention, for example, is that the
optical
bio-disc and disc reader assembly allows a person to carry out blood typing
analysis
in the field (that is, not in a clinical setting) expeditiously.
In various aspects of the invention, a sample is loaded into a chamber within
the optical bio-disc, having a capture field with one or more bioactive
capture agents
affixed thereto. The bio-disc is then subjected to conditions suitable for
cell binding.
Then, the bio-disc is placed into a CD drive assembly and is spun radially at
a speed
sufficient to separate unbound cells from bound cells, e.g., about 1000 rpm to
about
4000 rpm for about one to five minutes. This spinning causes the cells that
are not
bound by the capture agent to be removed from the capture fields and collected
in a
separate part of the chamber (e.g., in a waste receptacle in the chamber).
As used herein, the term "capture field" encompasses target zone or capture
zone 140 of an optical bio-disc, which has attached thereto, either directly
or
indirectly, a capture agent. The capture field is a discrete location on the
surface
having defined limits, metes and bounds.
As used herein, the term "capture agent" refers to any molecule A located on
the surface of the capture field that recognizes any molecule B, and binds
with
specificity thereto. The phrase "binds with specificity" is meant herein to
refer to the
binding of molecule A to molecule B by at least two fold, at least five fold,
at least 10
fold, at least one hundred fold, at least 1000 fold, at least 10,000 fold or
more when
compared to other molecules that may be present in a biological sample. By way
of
non-limiting example, molecules that specifically recognize and bind to other
molecules include antibodies, ligands, receptors, enzymes, substrates, biotin,
avidin,
and lectins. The bioactive agent of the invention may be obtained from any
source,
including but not limited to viral, bacterial, fungal, plant, animal, in vitro
or synthetically
produced materials.
In certain embodiments of the invention, the capture agent is a capture
antibody and the capture field has at least one capture antibody bound
thereto. As
used herein, the term "antibody" includes polyclonal, monoclonal, and
recombinantly
created antibodies. Antibodies of the invention can be produced in vivo or in
vitro.
Methods for the production of antibodies are well known to those skilled in
the art.
For example, see Antibody Production: Essential Techniaues, Peter Delves
(Ed.),
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John Wiley & Son Ltd, ISBN: 0471970107 (1997). Alternatively, antibodies may
be
obtained from commercial sources, e.g., Research Diagnostics Inc., Pleasant
Hill
Road, Flanders, NJ 07836 and Ortho Diagnostic Systems)
The selection of a capture agent to be bound to a capture field is within the
skill
of those in the art. By way of non-limiting example, a receptor-specific
ligand may be
bound to a capture field for the purpose of binding cells expressing the
receptor
recognized by the ligand or a capture field may be bound by a lectin that
binds
specifically a sugar moiety expressed on the surface of a select population of
cells for
the purpose of binding those cells. Alternatively, the capture field may be
bound by a
capture antibody specific for a receptor on the surface of a cell. Thus, the
invention
provided herein is easily adapted to any number of biological assays. Related
aspects regarding binding capture agents onto solid support, such as an
optical disc
substrate, is disclosed in, for example, commonly assigned U.S. Patent
Application
Serial No. 10/194,396 entitled " Multi-Purpose Optical Analysis Disc For
Conducting
Assays And Various Reporting Agents For Use Therewith" filed July 12, 2002,
which
is herein incorporated by reference in its entirety.
The term "antibody" is not meant to be limited to antibodies of any one
particular species, e.g., human, mouse, rat, goat, etc., are all contemplated
by the
invention. The term "antibody" is also inclusive of any class or subclass of
antibodies,
as all antibody types may be used to bring about an agglutination reaction. By
way of
non-limiting example, the IgG antibody class may be used for agglutination
purposes
or, if higher antibody polyvalency is desired, the IgM class of antibodies may
be
utilized for the same purpose. Other types of immunoglobulins that bind
specifically to
cells are also included within the scope of the invention. Antibody fragments
can also
be utilized as a capture agent of the invention. The use of antibodies in the
art of
medical diagnostics is well known to those skilled in the art. For example,
see
Diagnostic and Therapeutic Antibodies (Methods in Molecular Medicine), Andrew
J. T.
George and Catherine E. Urch (Eds.), Humana Press; ISBN: 0896037983 (2000) and
Antibodies in Diagnosis and Therapy: Technologies, Mechanisms and Clinical
Data
(Studies in Chemistry Series), Siegfried Matzku and Rolf A. Stahel (Eds.),
Harwood
Academic Pub.; ISBN: 9057023105 (1999), which are incorporated herein in their
entirely by reference.
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The capture field with the capture agent bound thereto can be structured in
any
way suitable to bind cells. In certain embodiments of the invention, one or
more
capture agents can be directly linked to the capture field. Thus, capture
fields may be
uniformly bound with multiple copies of a single capture agent or,
alternatively,
capture fields may be bound with multiple copies of two or more capture agents
to
increase the specificity of the binding reaction. In other embodiments, the
capture
agent can be indirectly linked to the capture field. By way of non-limiting
example, a
capture field may be coated with a protein such as streptavidin and a capture
agent
such as an antibody can be linked to the streptavidin by way of a biotin
moiety
attached to the antibody.
In certain embodiments of the invention, the capture field of the invention
has a
first capture agent bound thereto and the first capture agent binds a second
capture
agent. By way of non-limiting example, an anti-IgM IgG antibody can serve as a
first
capture agent bound to a capture field, which itself binds an IgM antibody,
the second
capture agent. Thus, the capture agent bound to a capture field can in certain
embodiments comprise more than one capture agent linked to one another in
tandem.
In various aspects of the invention, the capture agents may be attached to the
capture field in different ways. By way of non-limiting example, various
constructions
are presented in Table 2. Table 2 also demonstrates that different target
zones or
fields (i.e., windows) can be constructed with different capture agents on the
same or
different optical bio-discs. In one embodiment, a capture antibody can be
attached
directly to the bio-disc. In other embodiments, one or more additional agents
are
utilized as intermediate binding agents between the optical bio-disc and the
capture
agent located most distally thereto. This latter design reduces steric
hindrance,
thereby improving the likelihood that the capture agent will function
sufficiently.
Some embodiments depicted in Table 2 take advantage of the known strong
molecular interaction between streptavidin, or variants thereof, and biotin.
By way of
non-limiting example, streptavidin can be utilized as an initial layer in the
capture field.
By utilizing a biotinylated capture antibody, specific attachment to the
streptavidin
layer can be achieved by way of the molecular recognition between streptavidin
and
biotin and the strong binding resulting therefrom. If it is advantageous to
further limit
steric hindrance, a biotinylated first capture antibody can be bound to the
streptavidin
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layer, and a second capture antibody that is specifically recognized and bound
by the
biotinylated first antibody can be applied.
TABLE 2
Capture Layer Assembly and Variations
Window 1 2 3 4 5 6 7 8
OptionalStreet-Street-Street-Street-Street-Street-Street-Strept-
Initialavidin avidin avidin avidin avidin avidin avidin avidin
Layer
OptionalAnti-IgXAnti-IgXAnti-IgXAnti-IgXAnti-IgXAnti-IgXAnti-IgXAnti-IgX
1S' AntibodyAntibodyAntibodyAntibodyAntibodyAntibodyAntibodyAntibody
Capture
Antibody*
2"" Anti-A Anti-B Anti-D Anti-C Anti-c Anti-E Anti-a Anti-C""
CaptureAntibodyAntibodyAntibodyAntibodyAntibodyAntibodyAntibodyAntibody
Antibody
* Indicates that the capture antibody directly interacting with streptavidin
is biotinylated. IgX
refers to any antibody immunoglobulin, e.g., IgG, IgM, etc.
Referring now to Fig. 18, there is presented alternative capture technologies
of
the invention that are not reliant upon streptavidin/biotin interaction. As
shown in the
bottom row of Fig. 18, in the simplest embodiment a capture agent is attached
directly
to the optical bio-disc capture field. Capture agents include, but are not
limited to,
molecules such as IgG, IgM, a lectin, or other molecules. Because steric
hindrance
may prevent a capture agent linked directly to an optical bio-disc from
functioning
optimally, an alternative embodiment is to attach the capture agent to the bio-
disc by
way of an intermediate linker molecule (or cross-linker). Such cross-linkers
are known
in the art. By way of non-limiting example, any carbon compound having a
sufficient
number of carbon atoms to provide the requisite length to minimize or
eliminate
problems associated with steric hindrance will suffice.
It should be understood that a cross-linking system involves one or more cross-
linking agents, or conjugates, to cross-link one or more macromolecular
moieties to
another. A cross-link may be a covalent or non-covalent interaction between
two
macromolecular moieties, usually formed when two macromolecular free radicals
combine. Chemical modifications or conjugation processes to achieve cross-
links
involve the reaction of one functional group with another, resulting in the
formation of
a bond. The creation of bioconjugate reagents with reactive or selectively
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CA 02467740 2004-05-19
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functional groups forms the basis for simple and reproducible cross-linking or
tagging
of target molecules ("Bioconjugate Techniques," Greg T. Hermanson, Academic
Press, San Diego, CA, (1996)).
Cross-linking agents include, but are not limited to homobifunctional linkers,
heterobifunctional linkers, and zero-length cross-linkers. Homobifunctional
linkers are
linkers with two reactive sites of the same functionality, such as
glutaraldehyde.
These reagents could tie one protein to another by covalently reacting with
the same
common groups on both molecules. Heterobifunctional conjugation reagents
contain
two different reactive groups that can couple to two different functional
targets on
proteins and other macromolecules. For example, one part of a cross-linker may
contain an amine-reactive group, while another portion may consist of a
sulfhydryl-
reactive group. The result is the ability to direct the cross-linking reaction
to selected
parts of target molecules, thus garnering better control over the conjugation
process.
Zero-length cross-linkers mediate the conjugation of two molecules by forming
a bond
containing no additional atoms. Thus, one atom of a molecule is covalently
attached
to an atom of a second molecule with no intervening linker or spacer. One of
ordinary
skill in the art would refer to "Bioconjugate Techniques," Greg T. Hermanson,
Academic Press, San Diego, CA, (1996), for a detailed description of cross-
linking
agents.
In the present invention, cross-linking agents are bound to the surface of a
bio-
disc to immobilize capture agents within the target zones. A preferred cross-
linking
system is the heterobifunctional group consisting of biotin-streptavidin, i.e.
biotinylated
capture agents bound to an avidin-coupled substrate.
Other embodiments of the capture technology designed to reduce or eliminate
steric hindrance are presented in the middle and top row of Fig. 18. These
methodologies utilize either IgM or IgG as an intermediate binding molecule
between
the optical bio-disc and the capture agent. Capture agents include IgG, IgM, a
lectin,
or any other molecule that binds cells for the purpose of capture.
Referring to Fig. 19, cell capture technology of the invention based on the
strong recognition and binding of streptavidin (or variants thereof) and
biotin
molecules to each other is presented. As previously detailed in Table 2, the
bioactive
agent streptavidin can be first applied to the capture field and used to bind
and hold
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various capture agents, e.g., biotinylated IgG or biotinylated IgM or
biotinylated lectin
or any other cell binding molecule that is biotinylated. Various embodiments
following
this design are presented pictorially by the top row of Fig. 19. Alternative
embodiments are presented pictorially in the bottom row of Fig. 19, wherein
molecules
such as IgG or IgM are used as agents serving an intermediary function for the
binding of the aforementioned capture agents to a streptavidin layer on the
optical bio
disc. In these embodiments, the intermediary function served by IgG and IgM is
to
reduce or eliminate problems associated with steric hindrance, analogous to
the
function served by the linker molecule discussed in relation to capture
technologies
presented in Fig. 18.
An optical bio-disc of the invention can have multiple capture fields within
one
chamber. A grouping of several capture fields is termed a "bar code" because
the
data resulting from cells binding to certain capture fields resembles the dark
and light
striped pattern known as a bar code. In another example, also incorporated
within
such a bar code are defined negative control areas and positive control areas.
Referring now to Fig. 20, there are shown results of an exemplary assay
method of the invention for forward blood typing. The method utilizes multiple
capture
fields that are conveniently designed into a "bar code" format for sample
testing and
data presentation. In the embodiment presented in Fig. 20, the optical bio-
disc
contains several capture fields, each of which has affixed thereto a capture
agent,
e.g., antibody, which is specific for a particular ABO antigenic determinant
on the
surface of a red blood cell (anti-A and anti-B antibodies may be obtained from
Fisher
Scientific, Los Angles, CA, Catalogue Nos. 23287247 and 23287248,
respectively). In
addition, specific capture fields are designed as positive (POS) and negative
(NEG)
controls. Positive and negative controls for the test would include a positive
control
capture field in which the capture agent is a molecule that binds all cells,
e.g., a lectin
isolated from Lycopersicon esculentum that binds ~i-D-glucosamine oligomers
(Sigma
Aldrich Chemical, Catalog No. L-0651 ), and a negative control capture field
having no
capture agent bound thereto or a capture agent that is specific for another
molecule
that is not present in the sample.
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Bar code results pictorially presented in Fig. 20 demonstrate that data output
collected by means of standard microscopic analysis and data output collected
using
the CD imaging technology of the invention described herein are
indistinguishable.
A direct benefit of this type of approach is that the analysis of blood
samples
from subjects can then be compared to a known barcode result in order to
determine
the blood-type of that subject immediately. In the example presented, more A-
type
red blood cells are bound in the A capture field than B-type red blood cells
in the B
capture field, indicating that the individual being tested has an A red blood
cell
phenotype.
Various embodiments of this method of the invention may be similarly designed
for
the purpose of blood typing according to any other blood typing system, e.g.,
for
testing the Rh system blood group, the MNSs system blood group, the Lutheran
system blood group, the Kell system blood group, the Lewis system blood group,
the
Duffy system blood group, the Kidd system blood group, the Fisher system blood
group, or any other blood group. As will be understood by those in the art,
one or
more blood type systems may be simultaneously tested on a single bio-disc.
Antigentic Determinants
Various aspects of the present invention are drawn to methods for typing
blood. The
surfaces of red blood cells contain large numbers of antigenic determinants
that are
classified into blood groups. These antigenic determinants represent red blood
cell
surface markers that consist of protein andlor carbohydrate moieties. In
humans
there are at least 23 blood type groups (The Blood Group Antigen Factsbook
(Factsbooks Series) by Marion E. Reid (Editor) and Christine Lomas-Francis
(Editor)
(January 1997) Academic Press; ISBN: 0125859651 ). The ABO blood grouping is
perhaps the most important, serving as the basis for the determination of
transfusion
compatibility. Another frequently relied upon red blood cell grouping is the
Rhesus
(Rh) blood grouping, which is an important test during pregnancy.
A variety of other blood typing systems are amenable to the methods of the
invention. The most important of these include, but are not limited to, the
MNSs
System, the Lutheran System, the Kell System, the Lewis System, the Duffy
System,
and the Kidd System, the Fisher group, or another blood group. For a detailed
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discussion of blood transfusion technologies and the basis for blood group-
typing, the
following references are recommended: Transfusion Medicine by Jeffrey
McCullough
(December 1997), McGraw-Hill Professional Publishing; ISBN: 0070451133; Modern
Blood Banking and Transfusion Practices by Denise Harmening (Editor) (March
1999), F.A. Davis Co; ISBN: 080360419X; Immunohematolow: Principles and
Practice by Eva D. Quinley (Editor) (January 1998), Lippincott Williams &
Wilkins
Publishers; ISBN: 0397554699; and The Principles and Practice of Blood
Grouping by
Addine G. Erskine, ASIN: 0801615305.
Most blood typing tests are based on hemagglutination and involve mixing a
blood sample with a panel of typing reagents that react with various surface
antigens
and cause the cells to agglutinate. The presence or absence of agglutination
is an
indication of a specific blood type. The invention described herein utilizes a
cell-
capture technology uniquely adapted to a bio-disc format. The biological
assays of
the invention are designed to detect cell agglutination and/or cell binding.
In certain
embodiments of the invention, the subject for blood typing is a mammal, e.g.,
a
mouse or a human. In another example, the subject is a non-human mammal or a
non-human primate.
In certain embodiments of the invention, methods are provided for ABQ and/or
Rh typing of blood. The specific antibodies and antigens relevant for the ABQ
blood
typing system are presented in Table 3.
Thus, individuals whose red blood cells carry the A antigen have antibody in
their system directed against the B antigen, and individuals whose red blood
cells
carry the B antigen have antibody in their system directed against the A
antigen.
Individuals with both A and B antigens on their red blood cells produce no
antibody
directed against these antigens, and individuals with neither antigen present
on their
red blood cells have antibodies directed against both antigens in their
system.
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TABLE 3
The ABO Blood Tvae Svstem
Antigen Antibody Blood Group
A Anti-B A
B Anti-A B
A and B None AB
None anti-A, anti-B O
With the Rh blood typing system, there are three genes making up Rhesus
antigens: C, D, and E, all found on chromosome 1. If an individual's Rh
genotype
contains at least one of the C, D, E antigens, they are Rhesus positive. Only
individuals with the genotype cde/cde (rr) are Rhesus negative.
The remaining minor blood groups may complicate a blood typing but are not
as important. In an individual, antibodies to the antigens not expressed on
red blood
cells are non-red-cell stimulated (or naturally occurring), due to the
similarity between
the blood group antigens structure and environmental agents. The antibodies
may be
of the IgM, IgA or IgG class. The IgM antibodies can cause direct
agglutination when
mixed with cells bearing the antigen to which the antibody is directed
against. When
an individual is exposed to red cells that are incompatible with his or her
blood type,
either through a transfusion or through pregnancy, the individual's immune
system
produces antibodies against the introduced foreign blood type; the antibodies
produced by this process are predominately of the IgG class. The IgG
antibodies can
cross the placenta and cause hemolytic disease of the newborn in subsequent
pregnancies.
As stated above, the ABO and Rh blood groups are the most important groups
in transfusion medicine. Naturally occurring antibodies to the ABO and Rh
antigens
are of the IgM class. Antibodies to the ABO and Rh antigens are readily
available and
direct agglutination tests can be performed on red blood cell samples. In the
system
of the present invention, for forward typing assays, agglutination of the red
cells is not
the test; the test in this instance is cell capture based on antigen-antibody
interactions. The interaction is specific and accurate and indicates which
antigens are
present on the red blood cell, surface. For reverse typing of the ABO
antibodies,
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agglutination of cells captured on the optical disc is looked for and analyzed
by
software algorithm(s).
In general, the invention described herein encompasses three types of blood
cell typing. With respect to the ABO and Rh blood type systems, the assays
include
"forward" typing, in which antigens present on the surface of the red blood
cells are
detected by way of a cell capture assay, and "reverse" typing, in which
antibodies
directed to an ABO or Rh phenotype present in a patient's serum are detected
by a
cell agglutination assay. The third type of blood cell typing assay is
referred to as
"antibody" blood typing. It is a diagnostic assay designed to detect the
presence of
antibodies in a patient's serum directed to other, minor blood group
phenotypes, e.g.,
Kell, Duffy, Kidd, etc. In addition, the Rh blood type of an individual can be
determined with this type of assay. With antibody typing, the diagnostic test
is based
on cell capture and/or cell agglutination.
Forward Typing Assay
Figs. 21A to 21 F shows an example of a representation of the capture
chemistry of one embodiment of the forward typing assay on a reflective zone
disc.
More specifically, Fig. 21 A shows the substrate 120 coated with reflective
layer 142
and capture zones 140 where the reflective layer has been removed. The
reflective
layer may be removed by lithography, for example. Fig. 21 B shows a layer of
streptavidin 270 passively adsorbed to the capture zones 140. Fig. 21 C shows
biotinylated first capture antibody 272 bound to the steptavidin 270 in the
capture
zone 140. Fig. 21 D shows the second capture antibodies 274, 276 and 278 with
different specificities bound to the biotinylated first capture antibody 272.
Fig. 21 E
shows an assembled bio-disc with cap portion 116, reflective surface 146 and
inlet
port 122, adhesive member 118, channel 128 and the following capture
chemistries:
streptavidin 270, a first capture antibody 272, and three different second
capture
antibodies 274, 276 and 278 in the capture zone 140 on the substrate 120. Fig.
21 F
shows specific cell capture by capture antibody 274 based on antigens
expressed on
the surface of a cell. Capture antibodies 276 and 278 have no specificity of
binding
for cells being tested in the experiment presented. Fig. 21 F also
demonstrates the
method of detection by focusing an incident beam of electromagnetic radiation
152
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passing through a capture zone 140 to strike a reflective layer, thereby
producing a
return beam of electromagnetic radiation 154 which is delivered to a detector
system
157.
As used herein, the term "chamber" encompasses any three-dimensional
space defined by at least one material that is affixed to or part of an
optical bio-disc.
In one embodiment, the chamber is leak-proof so that a liquid sample may be
loaded
into the chamber and subjected to certain reaction conditions (such as
antibody
binding conditions) and to detection methods (such as beam interrogations).
The
chamber may be made of plastic, of metal, of glass or of any other material
that is
suitable for the biological assay for which the optical bio-disc is used. In
one non-
limiting example, the chamber may hold from about 4 NI to about 50 pl. In
another
example, the chamber is in fluid communication with a second chamber that can
be
utilized as a waste repository following the biological assay.
As used herein, an antibody that "specifically binds" means an antibody that
binds to an epitope, which comprises a peptide sequence, or a carbohydrate
moiety,
or a lipid moiety, or a specific sequence of oligonucleotides, or a
combination thereof.
Such an antibody will not promiscuously bind to other molecules that do not
have that
specific epitope. Such a specifically binding antibody will not bind (or cross
react) with
other molecules or compounds lacking such an epitope.
The assay is performed within an optical bio-disc that includes a chamber
(also, "flow chamber") having specific antibodies or other capture molecules
attached
to the solid phase associated with that chamber. In one non-limiting example
of the
invention, a method is described for the determination of the occurrence of a
specific
cell type (e.g., a specific type of red blood cell) expressing cell-specific
surface
antigens (e.g., A or B antigens) captured by specific antibodies affixed to
the capture
field(s).
An optical bio-disc drive assembly is employed to rotate the disc, read and
process any encoded information stored on the disc, and analyze the cell
capture
fields in the flow chamber of the bio-disc. The bio-disc drive is provided
with a motor
for rotating the bio-disc, a controller for controlling the rate of rotation
of the disc, a
processor for processing return signals from the disc, and analyzer for
analyzing the
processed signals. The rotation rate is variable and may be closely controlled
both as
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to speed and time of rotation. The bio-disc may also be utilized to write
information to
the bio-disc either before, during or after the assay. The test material in
the flow
chamber and capture fields is interrogated by the read beam of the drive and
analyzed by the analyzer. The bio-disc may include encoded information for
controlling the rotation of the disc, providing processing information
specific to the
type of immunotyping assay to be conducted and for displaying the results on a
monitor associated with the bio-drive.
The methods encompass evaluation tests in CD, CD-R, DVD, or any equivalent
optical disc format. Variations or alternative versions thereof according to
the present
invention include a robust capture chemistry that is stabilized on the optical
bio-disc.
Unbound non-specific cells are spun off leaving behind specific target cells
from the
blood sample that are specifically bound to the capture field on the bio-disc.
The read
or interrogation beam of the drive detects the captured cells and generates
images
that can be analyzed.
Reverse Typina Assay
The invention also provides a method for detecting specific antibodies to an
ABO/Rh blood group antigen, e.g., assaying a patient's serum for the
occurrence of
anti-A or anti-B antibodies. In one embodiment, the invention provides a
method for
reverse typing wherein the sample undergoes processing prior to being loaded
onto a
bio-disc (Figs. 22, 23, 24A, 24B, 24C, 25A, 25B, 25C, 26A, 26B, and 26C). In
another
embodiment, the invention provides a method for reverse typing wherein the
sample
is loaded onto a bio-disc without significant processing (Fig. 27).
Referring to Fig. 22, in the first embodiment of reverse typing, whole blood
is
first collected, e.g., by finger stick, and cells are separated from serum
prior to utilizing
the serum in the bio-disc blood grouping assay. Whole blood can be separated
into
serum and cells by light centrifugation, for example. The serum, which
contains a
patient's antibodies, is then mixed with one or more cell types having an ABO
cell
phenotype. For example, cell 1 of Fig. 22 can be an A type cell, cell 2 of
Fig. 22 can
be a B type cell, and cell 3 of Fig. 22 can be an AB type cell. The sample is
then
incubated for a period of time, e.g., about one to five minutes, at room
temperature to
allow the patient's antibodies to interact with these cells. After incubation,
if an anti-
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human antibody is used as the capture agent, the cells are washed several
times and
loaded into one or more chambers in the bio-disc; if a lectin capture agent is
used,
washing is unnecessary.
If antibodies of the appropriate specificity are found in the patient's serum,
the
red blood cells will be agglutinated as a result of the antibodies bound
thereto. These
agglutinated cells can then be captured on a capture field by an appropriate
capture
agent, e.g., a lectin that binds all cells. After a brief spin of the disc,
e.g., 400 rpm to
4000 rpm, to remove unbound cells, the capture field is examined for the
occurrence
of agglutinated cells. The capture field can then be examined by the optical
reader to
determine whether cells being tested are agglutinated, and thereby determine
that
antibodies to an ABO/Rh blood group antigen were present in the individual's
blood.
In Fig. 23 there is shown a second embodiment of reverse typing, whole blood
is first collected, e.g., by finger stick, and cells separated from serum
prior to utilizing
the serum in the bio-disc blood grouping assay. Whole blood can be separated
into
serum and cells by light centrifugation, for example. The Reagent Type A and
Type B
cells are mixed. Then the serum, which contains a patient's antibodies, is
added to
the cell mixture to thereby create a sample mixture. The sample mixture is
then
incubated for a period of time, e.g., about one to five minutes, at room
temperature to
allow the patient's antibodies to interact with these cells. After incubation
the assay
mixture is loaded into one chamber which has at least one anti-A capture zone
and
one anti-B capture zone. The capture of the agglutinated and non-agglutinated
cells
is illustrated and described below in conjunction with Figs. 26A, 26B, and
26C.
If antibodies of the appropriate specificity are found in the patient's serum,
the
red blood cells will be agglutinated as a result of the antibodies bound
thereto. If anti
A antibodies are present in the serum sample, for example, then agglutination
of the
Type A cells occurs. The agglutinated cells or un-reacted single cells are
then
captured on the specific capture zones. After a brief spin of the disc, e.g.,
400 rpm to
4000 rpm, to remove unbound cells, the capture zones are examined for the
presence
of agglutinated cells and single cells. The capture zones can then be examined
by
the optical reader to determine whether cells being tested are agglutinated or
single,
and thereby determine that antibodies to an ABOIRh blood group antigen were
present in the individual's blood.
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Referring now to Fig. 24A, there are shown test cells or typed reagent cells,
previously mixed with a patients serum, agglutinated by the patient's
antibody, loaded
into the inlet port 122 of an optical bio-disc having specific capture fields.
As can be
seen in the figure, at this stage, cells are agglutinated but not bound to a
capture
agent of the capture field. In Fig. 24B, after a pre-determined incubation
period, the
agglutinated test cells are specifically recognized and captured by the
capture agent
immobilized on the capture field. After a sufficient time for capture to occur
(incubation time), the optical bio-disc is spun and all cells not bound by the
capture
agent are removed from the capture field (Fig. 24C). Once unbound cells are
removed, data detection is accomplished by focusing an incident beam of
electromagnetic radiation 152 through the capture field to strike a reflective
layer,
thereby producing a return beam of electromagnetic radiation 154 that is
delivered to
a detector system 157. Analysis of the data provides information relating to
whether
the cells that bound to the capture agent were agglutinated or not
agglutinated.
In contrast, test cells that are not reactive with a patient's serum will not
be
agglutinated. Fig. 25A depicts test cells not agglutinated by a patient's
serum loaded
into an inlet port 122 of an optical bio-disc having a capture field. After a
sufficient
period of incubation, these cells are captured by the capture agent bound to
the
capture field (Fig. 25B). Once unbound cells are removed by centrifugation
(Fig.
25C), data collection and analysis are done as described for Fig. 24C. The
analysis
software of the invention can discriminate between single cells and
agglutinated cells
bound to the capture field.
Referring next to Fig. 26A, there is illustrated an assay solution containing
agglutinated and single cells being added to the chamber. The assay solution
may be
prepared as described above in Fig. 23. After a sufficient period of
incubation, the
agglutinated and non-agglutinated reagent cells are captured by their
respective
capture agents bound to the capture zone, as depicted in Fig. 26B.
Furthermore, Fig.
26B shows a capture zone with agglutinated cells and a separate capture zone
with
single cells. Once unbound cells are removed as shown in Fig. 26C, data
collection
and analysis are done as described for Fig. 24C. The analysis software of the
invention can discriminate between single cells and agglutinated cells bound
to the
capture field.
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In the third embodiment of reverse typing, whole blood, or a diluted sample
thereof, is loaded directly onto the bio-disc into a microfluidic circuit,
microfluidic
channel, or flow channel as illustrated in Fig. 27. The present method
provides for the
separation of blood cells and serum by passage through a separation chamber
250 in
the optical bio-disc. Separation of the fluid and cellular components of whole
blood is
effected by spinning the disc at a first speed, moving the sample through at
least one
microfilter designed to separate red blood cells, white blood cells and
platelets from
the serum. Serum is then moved to at least one mixing chamber 252 by spinning
the
disc at a second speed, which is higher than the first speed. Cells of a
specific ABO
group phenotype are then added through a separate entry port 256 into at least
one
mixing chamber 252. Mixing of the serum and cells is accomplished by spinning
the
disc at least once, one-half a rotation counter clockwise and then clockwise
one-half a
rotation. The samples are then allowed to incubate in the mixing chamber 252
for a
sufficient time to allow antibody-antigen interaction. The cells are then
moved to a
capture chamber or analysis chamber 254 with a capture field by spinning the
disc at
a third speed, which is higher than the second speed. The cells are allowed to
interact with the capture field which has bound to it anti-human
immunoglobulin or
another capture agent for a sufficient time to allow capture agent interaction
with the
cells. The disc is then spun again to remove unbound cells, e.g., 400 rpm to
4000
rpm. Data is then collected from the capture fields to determine if cells
bound thereto
are agglutinated. The occurrence of agglutinated cells in a capture field
indicates that
the individual's serum has antibodies directed to an antigen on the surface of
the
particular red blood cell blood type phenotype being tested. Alternatively,
the capture
chamber 254 may be packed with a bio-matrix that separates the agglutinated
cells
from the non-agglutinated cells. Details regarding this aspect of the
invention are
discussed below in conjunction with Figs. 35, 36A, 36B, 37A, 37B, and 37C.
Direct Typin Ag ssay_
In another aspect, the invention provides methods for antibody typing (or
direct
typing) a blood sample, i.e., testing a patient's serum for the occurrence of
antibodies
directed to an antigen of a blood group other than that of the ABO system. In
one
embodiment of this aspect, the invention provides a method for antibody typing
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wherein the sample undergoes processing prior to being loaded onto a bio-disc
(Figs.
28, 29A, 298, 29C, 30A, 30B, and 30C). In another embodiment of this aspect,
the
invention provides a method for antibody typing wherein the sample is loaded
onto a
bio-disc without significant processing (Fig. 31 ). Cells of a known blood
group
phenotype other than that of the ABO system, e.g., Kell, Duffy Kidd, etc., are
available
commercially for testing purposes (Immucor, Inc. Norcross, GA, PANOSCREEN).
Referring to Fig. 28, in yet another embodiment of antibody typing, whole
blood
is first separated from serum prior to utilizing the serum in the bio-disc
blood grouping
assay. Whole blood can be separated into serum and cells by light
centrifugation.
The serum, which contains a patient's antibodies, is then mixed with one or
more O
type ABO test cells having a known phenotype for a blood group type other than
that
of ABO. The sample is incubated for a period of time, e.g., about fifteen to
thirty
minutes, at 37° C to allow the patients antibodies to interact with
these cells. After
incubation, the cells are washed several times and loaded into one or more
chambers
in the bio-disc. If antibodies of the appropriate specificity are found in the
patient's
serum, the red blood cells will have the antibodies bound thereto. These
antibody-
bound cells can then be captured on a capture field by an appropriate capture
agent,
e.g., an anti-human IgG. After a brief spin of the disc (e.g., 400 rpm to 4000
rpm) to
remove unbound cells, the capture field is examined for the occurrence of
cells. The
capture field can then be examined by the optical reader to determine whether
cells
being tested are present in the capture field, and thereby determine the
antibody
status of the individual.
The molecular recognition events occurring during the assay are depicted in
Figs. 29 and 30. In Fig. 29A, test cells previously mixed with a patients
serum and
bound by antibodies contained therein are loaded into the inlet port 122 of an
optical
bio-disc having a capture field. In Fig. 29B, after the indicated incubation
period, test
cells bound by a patient's antibody are specifically recognized and captured
by the
anti-immunoglobulin antibody (i.e., capture agent) immobilized on the capture
field
140. Following a sufficient capture time, the optical bio-disc is spun and all
cells not
captured by the capture agent are removed from the capture field (Fig. 29C).
Once
unbound cells are removed, data detection is accomplished by focusing an
incident
beam of electromagnetic radiation 152 through the capture field to strike a
reflective
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layer, thereby producing a return beam of electromagnetic radiation 154, which
is
delivered to a detector system 157.
In contrast, test cells that are not reactive with a patient's serum will not
be
bound to a capture field. Fig. 30A depicts the loading of test cells not bound
by
antibodies of a patient's serum into inlet port 122 of an optical bio-disc.
Even after a
sufficient period of incubation, these cells are not captured by the capture
agents
bound to the capture field (Fig. 30B). The cells are removed from the capture
field
after a brief, low speed spin (Fig. 30C) and data detection is accomplished as
above.
Another embodiment of the antibody typing method of the invention involves
the use of an optical bio-disc having at least one microfluidic circuit. Fig.
31 is a
pictorial flow diagram illustrating the steps involved in this method. As
described in
Fig. 28, blood is collected and appropriately diluted in preparation for the
assay. The
test sample is loaded into an applicator for loading into the inlet port of an
optical bio-
disc. Initially, the sample containing cells and serum enters a separation
chamber
250 having microfilters which separate the cells from the serum. The serum is
then
moved into a mixing chamber by centrifugation and type reagent test cells are
added
thereto. After a sufficient period of time, the sample is then moved into a
capture
chamber and analysis subsequently occurs.
Fig. 32 presents embodiments of the microfluidic circuit described above, in
more detail. The sample is first loaded into an inlet port 251 and enters a
separation
chamber 250 in the optical bio-disc. Spinning the disc at a first speed,
thereby moving
the sample through at least one microfilter designed to separate red blood
cells, white
blood cells, and platelets from the serum, effectively separating the fluidic
and cellular
components of whole blood. Serum is then moved to at least one mixing chamber
252 by spinning the disc at a second speed, which is higher than the first
speed.
Reagent cells of a known blood group phenotype are then added through a
separate
entry port 256 into at least one mixing chamber 252 of the bio-disc.
With various aspects and embodiments of the invention, the microfluidic
circuit
has one or more inlet ports 256 for the addition of cells of a known blood
group
phenotype (typed reagent cells) to the mixing chamber(s). In other various
aspects
and embodiments of the invention, the microfluidic circuit has a single mixing
chamber
feeding a single capture chamber 254. In yet other various aspects and
embodiments
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of the invention, the inlet ports) 256 are not necessary, since the mixing
chamber has
been preloaded with a microparticle coated with a specific antigen of a red
blood cell
blood type group, e.g., M antigen or N antigen. Such antigens may be
conveniently
prepared by purifying the red blood cell antigen, e.g., through recombinant
gene
expression, subsequent biochemical isolation, and absorbing it onto the
particles.
These particles may then be loaded into the mixing chamber during construction
of
the bio-disc, e.g., in freeze-dried form. A bio-disc of this construction
would be
particularly useful in countries and areas where access to red blood cells of
a known
blood type phenotype is difficult.
Mixing of the serum and cells is accomplished by spinning the disc at least
once one-half of a rotation counter clockwise and then clockwise one-half of a
rotation. The samples are then allowed to incubate in the mixing chamber 252
for a
sufficient time (e.g., 15 to 30 minutes) to allow antibody-antigen
interaction. The cells
are then moved to a capture chamber 254 with a capture field by spinning the
disc at
a third speed, which is higher than the second speed. The cells are allowed to
interact with the capture field, which has bound to it anti-human IgG, for a
sufficient
time (e.g., 30 seconds to 15 minutes) to allow antibody-antigen interaction.
The disc
is then spun (e.g., 400 rpm-4000 rpm) to remove unbound cells. Data is then
collected from the capture fields to determine if cells are bound to the
capture field.
The occurrence of cells in a capture field indicates that the individual's
serum has
antibodies directed to an antigen on the surface of the particular red blood
cell being
tested. An alternative embodiment of the above discussed antibody typing using
bio-
matrix cell or particle separation is described below in conjunction with
Figs. 35, 36A,
36B, 37A, 37B, and 37C.
Software and Related Processing Methods
Computer-based analysis is performed in a determination of blood type.
Results of executing a procedure involving an optical bio-disc are analyzed by
software to determine a blood type and/or antibody types of a blood sample
supplied
on the bio-disc.
In one or more specific implementations, the procedure involving the bio-disc
may be executed under software control. For example, software may prompt a
user
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to prepare the bio-disc (e.g., by loading a blood sample into the bio-disc and
inserting
the bio-disc into a reader), and may subject the bio-disc to one or more spin
sessions
at one or more rotation speeds. In a specific implementation, the procedure
may
include washing and/or incubation.
In one or more implementations, the software may cause execution of the
procedure to be responsive to input from the user or to intermediate results
of
executing the procedure, or to both. For example, the software may pause
execution
pending a signal (e.g., by keyboard or mouse) reporting that the user has
added
material to the bio-disc. In another example, the software may determine that
a
condition (e.g., the presence or absence of material in a particular location)
on the
bio-disc has been detected, and may cause the procedure to execute in a
particular
way based on the detection. In a specific implementation, the software may
determine a confidence level (e.g., based on a margin of error) of a
determination as
to blood type, and based on the confidence level the software may cause the
bio-disc
to be spun at a rate and a duration that delivers all or a portion of a blood
sample to a
microfluidic circuit or assay zone for another blood type determination.
In one or more implementations, the bio-disc may have write-compatible
properties (e.g., write properties of a CD-R or CD-RW) and the software may
cause
information (e.g., representing or derived from intermediate or final results
of the
procedure) to be written to the bio-disc and thereby made available for
subsequent
retrieval by the software or another program. The retrieval, which may occur
one or
more times, may occur during the same instance of execution of the procedure,
and
may thereby affect the execution of the remainder of the current instance, or
may
occur after or at the end of the current instance. For example, once the
software has
determined the blood type of the sample, information including an
identification of the
blood type and optionally underlying data as well may be recorded on the bio-
disc. In
such a case, in at least some circumstances, the bio-disc can be archived
(e.g., for
evidentiary or confirmation purposes in legal proceedings) so that the
software's
determination is available in a fixed form physically associated with the
actual sample
from which the blood type determination was made. The bio-disc can thus serve
as
an enhanced medical record of blood-typing, and can be retrieved later for
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analysis (e.g., DNA testing or direct year-to-year comparisons or cumulative
or
historical analysis under software control).
Accordingly, interactivity may be provided in one or more of at least two
forms:
interaction with the user and interaction with the bio-disc. Interaction
between the
software and the user may take the form of output to the user (e.g., on an
electronic
display, via voice or other audio, or via another mechanism that is detectable
by a
human sense such as smell or touch) and input from the user (e.g., via
keyboard,
mouse, joystick, microphone, light sensor, or another mechanism that allows a
computer to detect a physical change).
In one or more specific implementations, the results of the procedure may be
communicated in the form of one or more electronic signals that may be
produced
with the use of one or both of a moving laser head and a moving light detector
applied
to an optical bio-disc having one or more tracks, which achieves track by
track
generation of signal information. For example, analog signals representing
reflected
or transmitted light readings from an optical bio-disc may be received and may
be
converted to digital signals that are analyzed in the blood or antibody type
determination. As described in more detail below, the software may analyze the
electronic signals to identify, locate, and/or quantify transitions in light
intensity that
represent the boundaries of material (e.g., producing dark spots) resulting
from
execution of the procedure, and may draw a conclusion as to blood or antibody
type
based on the existence/absence, location, and/or quantity of the material.
The software and procedure may include or rely on an ABO technique or ABO
cell counting technique, or an antibody typing process that may include wash
and/or
preparation steps in microfluidic circuits and the moving of material to a
testing area
and/or a waste area for detection, e.g., by a light intensity and/or optical
density
technique.
With reference again to Fig. 32, there is illustrated an example of a
microfluidic
circuit that may be used with the software and/or the procedure. The software
may
control the timing of the use of areas 250, 252, 254, may cause information
such as
intermediate results to be recorded on the circuit's bio-disc, and may cause
execution
of the procedure to be affected by results or conditions found pertaining to
one or
more of, the areas 250, 252, 254. For example, the software may cause the bio-
disc
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to have a behavior A if a condition X is found in area 250, and may cause the
bio-disc
to have a behavior B if a condition Y is found in area 250. Similar tests and
actions
may be executed with respect to other areas of the bio-disc in addition or
instead. The
software can implement a branching process or a flowchart that is responsive
to
intermediate results or conditions found. Accordingly, different variations of
a blood
typing test, or different blood typing tests, can be implemented using the
same bio-
disc arrangement with correspondingly different variations or configurations
of
software. Depending on the test variation or test that is in effect, the
software can
cause the bio-disc to spin faster or slower at different times and with breaks
of
different durations, and can direct the drive's laser to expose different
areas to
different amounts of light, to create the procedural environment that suits
the test
variation or test.
In general, the software can implement interactive, multi-route processing
with
the bio-disc. For example, a microfluidic circuit may have 5-6 stages and the
software
may take certain action depending on a result in the first stage, and then may
take
other action depending on a result in the second stage, and may continue
similarly in
the remaining stages. The functionality of the bio-disc and other hardware is
thus
directed by the software and the results.
The software may treat conditions found on the bio-disc as abstractions such
as Event Capture types. For example, a glucose finding may be treated as Event
Capture 1, and genetically modified organism (GMO) testing may be treated as
Event
Capture 3. One or more parameter tables may be used to which software code is
directed and in which specific tests are implemented (optionally along with
interactivity
instructions). In development of tests, such tables accelerate time to market
and
reduce software code maintenance overhead. One parameter in such tables may
relate to event counting as described below. As shown in Fig. 34, other
parameters
may implement decision blocks of flowcharts representing blood-typing tests,
e.g., so
that a decision made pertaining to zone A causes an action to be taken in zone
B
instead of in zone C.
As described below, the software and bio-disc system may produce
quantitative as well as qualitative blood-typing results. For example, numeric
results
may be achieved that not only indicate a Type A blood, but also may
differentiate
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between a strong Type A and a weak Type A blood (e.g., 85% versus 15% Type A).
By supplying sensitivity, selectivity, and non-specific binding,
quantitatively enhanced
results are made possible.
Referring next to Fig. 33, there is shown a sample display image showing the
results of a blood typing assay performed using the methods and apparatus of
the
present invention. As indicated by the bar graph and corresponding actual
physical
counts of captured red blood cells, the blood sample is a strong Type A. Also
shown
are positive and negative control results for heightened confidence in assay
reliability.
In the case of a weak Type A, the corresponding bar may be much lower (e.g.,
just
above a threshold value for Type A). Thus, the software can quantitatively
detect the
strength of a blood type determination, and in certain circumstances, can
perform
error checking and/or redundant checking functions. Such functions include,
but are
not limited to, retesting in the case of a weak determination, changing a
threshold
value for a Type determination, applying a different test, using a different
sample size,
and requesting action by the user. Therefore, the output of the software will
be
produced only after the software has determined a blood type result to a
sufficient
confidence level.
Under software control, the bio-disc can thus perform sophisticated
interactive
blood typing analysis on relatively small blood samples, without having to
retrieve
further blood from a patient, which may be difficult or impossible or have
adverse
health consequences.
Other information technology resources can be applied to the bio-disc in the
blood typing techniques. For example, results of the software's analysis can
be
transmitted (e.g., in secure form) over a network such as the Internet for
storage or
further analysis. The software can be executed, updated, and/or maintained
over a
network such as the Internet. Reporting can be accomplished automatically by
triggering a communication, e.g., to a health agency or a demography-related
institution whenever a particular result is found during blood typing.
In a specific implementation, an event counting technique is used to detect
and
quantify material on the bio-disc and a blood typing result is produced based
on an
analysis of the results of executing the event counting technique.
Effectively, a track
by-track scan is made of a portion of a bio-disc, spots of material are
detected in the
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scan, and the blood type of a blood sample is determined and reported based on
the
quantity of detected spots.
In the technique, a sampling rate and optionally a bio-disc rotation speed is
adjusted to correspond to the expected or detected size of the spots so that
the spots
can be reliably counted in real time using conventional and practical
computing
resources. For example, if red blood cells of seven micron size are being
detected, a
sampling rate of 667 kilohertz at a 4x rotation speed may be used to detect
seven
micron spots while filtering out spots of significantly different sizes.
Event counting can be performed per track or per set of tracks, optionally in
real time. In a specific implementation, the read head of the optical drive
mechanism
is positioned at a designated track, and a set (e.g., a track's or a block's
worth) of
samples are read into memory. In the capture zone, an event is recorded when
the
readings match a predetermined profile, e.g., a transition such as a specified
number
of low intensity samples followed by a specified number of high intensity
samples.
Low intensity and high intensity may be treated as relative sample-to-sample.
Events
are recorded for all samples corresponding to the width of the capture zone
along the
track. The rest of the capture zone is scanned similarly on other tracks above
and/or
below the current track to locate spots that are not intersected by the
current track.
The event represents the spot, such that counting the events in the capture
zone
effectively produces a count of the spots in the capture zone.
In other embodiments, signal analysis such as digital signal processing may be
performed (e.g., to implement low pass or band pass filtering) to expose
features of
interest such as the spots of specified size that are counted. The laser
wavelength
and/or laser spot size may be altered to facilitate exposure of the features.
The blood typing analysis performed by the software may depend on the
number of spots (e.g., representing red blood cells) found in one or more
capture
zones such as target zones 140.
In a particular implementation, the event counting is performed in real time
as
the track data is being retrieved from the bio-disc, which allows the analysis
to be
performed with a small and inexpensive amount of memory, since all that is
retained
by the software is the event count itself, which requires little memory.
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In a specific implementation, an alignment mark is pre-disposed on the bio-
disc
specifically to allow the software to determine a starting point on the track
from which
to determine an offset point for the capture zone of interest. The alignment
symbol
may include a solid black India ink dot positioned near the start of a capture
zone.
The samples may represent voltage levels corresponding to light intensities
measured by a detector at the bio-disc. For example, a particular 14-bit
sample (or
upper 8 bits of a 14-bit sample) may correspond to undisturbed reflected or
transmitted light representing an absence of a feature of interest such as a
red blood
cell, and another sample may correspond to partially or entirely blocked light
representing the presence of the feature.
In a case in which the drive mechanism is derived from a conventional CD-
ROM drive, the software may use a logical block address (LBA) command to
direct
the mechanism to position the read head to read data from a particular track
of the
bio-disc. To specify a particular track, the track number is multiplied by 75
to produce
a logical block number and the drive is commanded to go to the logical block
number.
The electronic signals from the samples that are taken may include one or more
of
conventional CD-ROM analog signals A-D, HF1-HF2, Focus, and Tracking. The
samples may be derived from the analog signals by a Eultrad EDDA1280 A/D
device
from AIterView that can sample in a range from 400 kilohertz to 80 megahertz.
Other conventional CD-ROM commands that may be used by the software
include initialize drive, set drive speed, track out to location, track out to
location or LV
positioning, test burn mode, and drive reset.
The software may attempt up to a predetermined number of times (e.g., 12) to
find the alignment symbol before giving up. Multiple capture zones may be
positioned
along a particular track, with different offset points from the end of the
alignment
symbol. If the alignment symbol is found at the end of a block of data, the
data may
be discarded and another block may be read so that offsets may be calculated
properly from the end of the alignment symbol at a point closer to the
beginning of the
block.
As shown in Fig. 33, the output includes a bar graph and number counts
corresponding to the bars. Below the counts are boxes having spots, which are
effectively images of portions of capture areas. The images are formed line by
line as
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event counting is performed for tracks covering a capture area. A blood type
is
determined by calculating a ratio of counts in various capture areas.
The selection of a sample rate can be important: if the sampling rate is too
low,
the software may miss an object; if the sampling rate is too high, small
objects of little
or no interest may be improperly counted along with the objects of interest,
and the
data rate may become burdensome on the computing platform.
The computing platform may include a conventional personal computer
including but not limited to a 433 megahertz Pentium-compatible microprocessor
and
running Microsoft Windows 98, and the software may be implemented in a high-
level
language such as Microsoft Visual C++ or Visual Basic with assembly language
components where execution speed makes a significant difference (e.g., in real-
time
analysis of the samples).
Image analysis may be performed on the images of the capture areas to
expose additional features beyond counts, such as positional arrangements of
objects
and local and global densities of objects.
At least some of the techniques described above can be applied to platforms
other than that of microfluidic circuits on bio-discs.
Fig. 34 illustrates a high-level procedure that is executed by an
implementation
of the software. The procedure is used to allow parameters to be defined for
use in a
test such as a blood-typing test applied to a bio-disc.
Blood Analysis Using Bio-Matrix Separation
The hematology and immunohematology analyses methods and apparatus
described above in conjunction with Figs. 1 to 34 may be implemented using a
bio-
matrix method. The bio-matrix method is described above in detail in
connection with
the fourth aspect of the present invention. In this method, a bio-matrix is
formed in a
microfluidic channel or circuit in an optical bio-disc. The bio-matrix may be
formed
from cross-linked polymers or microparticles including polyacrylamide,
agarose, and
polstyrene or glass beads such as those used in liquid chromatography. Fig. 35
illustrates a micro-fluidic circuit 304 of an optical bio-disc containing a
bio-matrix 300
formed therein. The fluidic circuit 304 may also contain an assay solution
302. The
assay solution 302 may contain a buffer, reagent antibodies, or typed reagent
cells.
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Referring to Figs. 36A and 36B, there is illustrated the addition and reaction
of
particles or cells within a channel or microfluidic circuit. More
specifically, Fig. 36A
shows the addition of particles or cells into the bio-matrix microfluidic
circuit. The cells
may include typed reagent cells 306 or sample red blood cells 308. The
particles then
react with reagents in the assay solution to form agglutinates 310 shown in
Fig. 36B.
Once the agglutination reaction is completed, the disc is spun at a pre-
determined
speed and time to separate the agglutinated cells 310 from the non-
agglutinated cells
312 using the bio-matrix 300. The result may be any one of the patterns shown
in
Figs. 37A, 37B, or 37C. Fig. 37A depicts results from a strong agglutination
reaction
wherein all the particles or cells form agglutinates 310. The agglutinates 310
are
unable to enter the bio-matrix since the pores of the matrix are too small to
permit
passage of these agglutinates 310. In some cases, the agglutination reaction
is weak
such that the agglutinates formed 310 are small enough that they may enter the
matrix and are trapped within it as shown in Fig. 37B. If the analyte of
interest is not
present in the sample, agglutination does not occur and all the single, non-
agglutinated particles or cells 312 pass through the matrix and form a pellet
at the
bottom or distal end of the microfluidic circuit as shown in Fig. 37C. The
location and
amount of the agglutinates 310 and/or non-agglutinated cells 312 may then be
analyzed using an optical disc reader such as that described in the above
referenced
and incorporated U.S. Patent Application Serial No. 101043,688. Data from
these
analyses may then be interpreted using accompanying computer software and an
appropriate antigram incorporated into the disc and/or drive software to
generate a
blood type panel, and/or an antibody panel or profile. This method automates
the
blood typing and antibody screening process and decreases turn around time for
hematology and immunohematology testing.
In one embodiment of the present invention, separate microfluidic channels are
pre-loaded with reagent antibodies including anti-A, anti-B, or anti-AB
antibodies.
Diluted or washed patient or sample red blood cells are then added to each of
the
chambers to form an assay solution as shown in Fig. 36A. After a pre-
determined
incubation time, agglutination of the red blood cells, as shown in Fig. 36B,
occurs in
the channel containing the antibody that binds to the specific antigens (A, B,
or AB
antigens) on the red cells. For example, if the sample red blood cells are
type A then
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agglutination occurs in the channel containing anti-A antibodies. Once the
agglutination reaction is completed, the disc is spun at a pre-determined
speed and
time to separate the agglutinated cells from the non-agglutinated cells. The
result
may be any one of the patterns shown in Figs. 37A, 37B, or 37C. The most
common
results using this embodiment are presented in Figs. 37A and 37C which,
respectively, show a strong reaction to the appropriate reagent antibody and
no
reaction to other antibodies. Fig. 37B illustrates the less common weak
positive test
result.
In another embodiment of the present invention, separate microfluidic channels
are loaded with typed reagent cells including A, B, or O type reagent cells.
An aliquot
of patient serum or plasma is then added to the individual chambers to form an
assay
solution as shown in Fig. 36A. After a pre-determined incubation time,
agglutination
of the reagent cells, shown in Fig. 36B, occurs in the chamber containing the
appropriate type of reagent cells. For example, if the serum sample contains
antibodies for B type red blood cells (from a type A blood sample)
agglutination will be
observed in the channel containing B type reagent cells. Once the
agglutination
reaction is completed, the disc is spun at a pre-determined speed and time to
separate the agglutinated cells from the non-agglutinated cells. The result
may be
any one of the patterns shown in Figs. 37A, 37B, or 37C. Typical common
results
using this embodiment are shown in Figs. 37A and 37C which, respectively, show
a
strong reaction of the serum antibodies to the appropriate reagent cell type
and no
reaction to other cell types. Fig. 37B illustrates the less common weak
positive test
result.
In yet another embodiment of the present invention, separate microfluidic
channels are pre-loaded with Coomb's serum, or anti human globulin with or
without
complement. A serum sample is prepared and mixed individually with different
type O
reagent cells expressing different patterns of minor antigens including, but
not limited
to, the various types of Kell, MNSs, Duffy, Kidd, Lewis, and Lutheran
antigens, to form
different assay solutions. The different assay solutions may be loaded into
different
microfluidic channels in an optical disc as shown in Fig. 36A. Alternatively,
an aliquot
of the serum samples may be added to multiple chambers followed by the
addition of
the different type O reagent cells into different chambers containing the
serum
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sample. Antibodies present in the sample will specifically bind to their
respective
antigens expressed on the reagent cell membranes. The anti-human globulin
(AHG)
or Coomb's serum facilitates agglutination of the antibody coated red blood
cells by
binding to the antibodies bound to the cells and cross linking antibodies
bound to
other cells in the assay solution. If there are no antibodies present in the
sample
which have specific affinity to the target antigens expressed on the surface
of a
reagent cell, then agglutination of cells does not occur since the AHG cannot
cross
link cells without antibodies attached to the cell surface. After a pre-
determined
incubation time, agglutination of the reagent cells, shown in Fig. 36B, occurs
in the
chamber containing the appropriate type of reagent cells. For example, if the
serum
sample contains antibodies for k type red blood cells, agglutination will be
observed in
the channel containing reagent cells expressing the k antigen. Once the
agglutination
reaction is completed, the disc is spun at a pre-determined speed and time to
separate the agglutinated cells from the non-agglutinated cells. The result
may be
any one of the patterns shown in Figs. 37A, 37B, or 37C. Expected common
results
using this embodiment are shown in Figs. 37A and 37C which, respectively, show
a
strong reaction of the serum antibodies to the reagent cell types expressing
the
specific antigens of interest and no reaction to other cell types. Fig. 37B
illustrates the
less common weak positive test result. By incorporating the antigram for the
reagent
cells into a computer software analysis program, the patient's antibody
expression
pattern or antibodies present in the sample may be automatically determined
based
on the pattern of positive and negative reactions.
Referring to Fig. 38 there shown a top plan view of another embodiment of a
transmissive optical bio-disc 110 showing semi-circular, equi-radial fluidic
circuits 320
having inlet ports 122, vent ports 124, and trigger marks 126. The equi-radial
fluidic
circuits 320 include a semi-circular or arcuate analysis chamber that is
substantially
directed along an arc segment of an annular ring within the substrate 120 of
the
optical bio-disc 110. The capture zones 140 are placed within the semi-
circular
analysis chamber of the equi-radial fluidic circuit 320. Also shown in Fig. 38
are
trigger markings 126. The disc essentially includes all the components of the
transmissive optical bio-disc as described above in conjunction with Figs. 5,
6, 7, 8,
and 9.
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Referring next to Fig. 39 there is illustrated an enlarged detailed view of a
portion of the equi-radial fluidic circuit of the disc shown in Fig. 38. This
particular
fluidic circuit includes capture zones for the forward blood typing assay as
described
above in conjunction with Figs. 21 A to 21 F. As shown in Fig. 39, the
analysis
chamber may have, for example, several capture zones including, but not
limited to,
anti-A, anti-B, anti-H, anti-C, anti-c, anti-D, anti-E, anti-e, and Rh control
capture
zones for testing or determining the major blood group of a blood sample.
Other
capture zones with capture agents specific for other antigens can be
substituted for
any of the above mentioned capture zones.
With reference now to Fig. 40 there is depicted an enlarged detailed view of
yet
another embodiment of the transmissive disc with a proximal equi-radial
fluidic circuit
324 and a distal equi-radial fluidic circuit 322. The distal equi-radial
fluidic circuit 322,
in this embodiment, is used for forward blood typing as discussed in above
Fig. 39
while the proximal equi-radial fluidic circuit 324 is used for reverse blood
typing. The
reverse blood typing test serves as a confirmatory test for the result
generated from
the forward blood typing. Details relating to sample preparation for reverse
blood
typing and assay procedures are discussed above in conjunction with Fig. 23,
26A,
26B, and 26C, for example. In use, red blood cells are processed as described
below
in Example 2. The red blood cells are then loaded in to the distal equi-radial
fluidic
circuit 322 though the inlet port 122. Meanwhile, a blood sample is also
prepared for
reverse typing as described above in conjunction with Fig. 23 where the serum
or
plasma is mixed and incubated with Type A and Type B reagent cells. Then the
suspension of cells and serum is loaded into the proximal equi-radial fluidic
circuit 324
through the inlet port 122. The inlet and vent ports are then sealed and the
disc is
loaded into the optical disc drive 122 (Fig. 17) for analysis. During
analysis, the disc
may be rotated at a pre-determined speed and duration to remove cells or
agglutinates that are not bound by the capture agents in the capture zones.
The
capture zones in the distal fluidic circuit 322 are then investigated to
determine the
zones with bound cells. The capture zones in the proximal fluidic circuit 324
are also
investigated to determine which zone or zones have agglutinated cells or non-
agglutinated cells bound thereto (Fig. 26C). The forward and reverse typing
performed together is advantageous for accurately determining the blood type
of an
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individual. In standard clinical blood typing, the reverse typing serves as
confirmation
of the correct forward typing for ABO blood grouping. The present invention
permits
both the forward and reverse typing to be run concurrently with reduced sample
volume and minimal user intervention. In addition, the forward and reverse
typing
tests ofe the present invention may be analysed in approximately 2 minutes
using
appropriate hardware and software. Further details relating to optical disc
drives,
detection systems, and software that may be used in conjunction with the
optical bio-
discs of the present invention is disclosed in for example, commonly assigned
and co-
pending U.S. Patent Application Serial No. 10/241,512 entitled "Methods for
Differential Cell Counts Including Related Apparatus and Software Performing
Same"
filed September 11, 2002; and U.S. Patent Application Serial No. 10/279,677
entitled
"Segmented Area Detector for Biodrive and Methods Relating Thereto" filed
October
24, 2002; both of which are incorporated by reference in their entireties as
if fully
repeated herein.
Experimental Examples
While this invention has been described in detail with reference to the
drawing
figures, certain examples and further illustrations of the invention are
presented
below.
EXAMPLE 1
Bio-Disc and Capture Layer Preparation
In one embodiment, the tracking of the bio-disc of the present invention is a
forward Wobble Set FDL21:13707 or FDL21:1270 coating with 60 nm of gold. On
this
reflective disc, oval data windows of size 2 x 1 mm are etched out by
Lithography. "U"
shaped channels are used to create chambers that are 25 um in height. It takes
about 7 uls of sample to fill the entire chamber including the inlet and
outlet ports. A
8-window/4-channel format to be preferentially used. In the preferred
embodiment of
the invention a semi-reflective transmissive disc (FDL 20/21:00708) is used
which
allows the entire surface of a transmissive disc to be used for capture zones,
without
the use of lithography to form data windows. Fraylock "U" shaped adhesive DBL
201
Rev C 3M94661 or straight channels are used to create the chambers. The cover
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disc utilized is a gold disc, fully reflective with 48 sample inlets with a
diameter of
0.040 inches location equidistant at radius 26mm or a clear disc to allow use
of a top
detector.
Several chemical layers are applied sequentially to the solid substrate first
layer. This first layer may be a polycarbonate layer or a metalized
polycarbonate layer
in a optical disc such as a CD, CD-ROM, DVD or DVD-ROM. Prior to subsequent
treatment, the first layer is cleaned with isopropanol. The second layer
consists of
either polystyrene or polycarbonate. This layer may be formed by injection
molding of
bulk plastic or spin or spray coating of the plastic in a volatile solvent on
a solid
substrate.
The primary capture layer, the third layer, is formed by absorption of the
protein
streptavidin (Sigma, St. Louis, MO, Catalogue No. S-4762) (or any variant
thereof) on
the second layer. The adsorption process is accomplished by exposure of the
second
layer to a first solution (1 mg/ml solution of streptavidin (or any variant
thereof) at
neutral pH (+/- 0.5 pH) in either phosphate (sodium or potassium) or Tris
buffer, ionic
strength (varied by addition of NaCI, KCI or MgCl2) between 50 and 200 mM).
Exposure times may range between 30 seconds and 12 hours. After exposure of
the
second layer to the first solution, the excess streptavidin (or any variant
thereof) is
washed away with water.
The secondary capture layer, the fourth layer, consists of biotin-labeled
antibody (the first capture antibody) that recognize and bond to other
antibodies from
a certain animal source (e.g., mouse or human) (e.g., biotinylated anti-mouse
IgM
(raised in sheep), Vector Laboratories, Catalog # BA-2020). A solution of the
first
capture antibody (the second solution) is exposed to the third layer for
between 10
minutes and 3 hours. The second solution comprises a 0.5 mg/ml solution of the
first
capture antibody at neutral pH (+/- 0.5 pH) in either phosphate (sodium or
potassium)
or Tris buffer, ionic strength (varied by addition of NaCI, KCI or MgCl2)
between 50
and 200 mM. The biotin moiety on the surface of the first capture antibody is
bound
by the streptavidin (or any variant thereof), which comprises the third layer.
After
exposure of this layer to the second solution, the excess first capture
antibody is
washed away with water.
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The bioactive capture layer, the fifth layer, consists of the second capture
antibody, which recognizes and binds to a specific type of biological cell
based on
some antigen on the surface of that cell. The animal source of the second
capture
antibody must match the specificity of the first capture antibody. A solution
of the
second capture antibody is exposed to the fourth layer for between 10 minutes
and 3
hours. This third solution comprises a solution (possibly 1 mg/ml) of the
second
capture antibody at neutral pH (+/- 0.5 pH) in either phosphate (sodium or
potassium)
or Tris buffer, ionic strength (varied by addition of NaCI, ICCI or MgCl2)
between 50
and 200 mM. After exposure of the fourth layer to the third solution, excess
second
capture antibody is washed away with a buffer similar to that described above.
Since blood is analyzed, the discs of the invention are leak checked to make
certain that none of the chambers leak during spinning of the disc with the
sample in
situ. Each channel is filled with a blocking agent. Blocking is done for least
1 hour.
The discs are then spun at 5000 rpm for 5 minutes and examined. After checking
for
leaks and removing the blocking solution, the disc is placed in a vacuum
chamber for
2-48 hours. After vacuum treatment, discs are placed in a vacuum pouch and
stored
at 2-8°C until use.
Additionally, the disc can be heat or ultrasonically bonded to make certain no
fluid escapes from the chamber.
EXAMPLE 2
Forward Blood Typing Assay on Bio-Disc
In the example to follow, the forward blood typing assay is conducted on a bio-
disc comprising: (1 ) gold reflective base disc, treated with photo-
lithography to remove
the gold in specific capture zones, with appropriate chemistry placed over the
capture
zones, (2) 25 um thick channel layer, and (3) gold reflective cover disc,
assembled
into a functional bio-disc.
10 ul of whole blood from a finger stick is diluted in 90 ul of phosphate
buffered
saline/anticoagulatant to make a 10% RBC solution. 14 ul of this is
injected'into the
functional bio-disc and the inlet and vent ports are sealed. After a five-
minute room
temperature incubation, the disc is placed into the drive. The automated event
counting software developed in-house centrifuges the disc, causing the non-
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specifically captured cells to be removed from the capture zones. The disc is
scanned
with the standard 780 nm laser of the optical drive using the bottom detector
and the
software registers the number of events in each capture zone. The program
algorithm
determines which capture zones had a positive capture and assigns an ABO and
Rh
phenotype to the blood sample. The entire process takes about 10 to 15 minutes
from
insertion of disc into the drive and receiving the forward blood typing. The
diagnostic
protocol is presented pictorially in Fig. 8.
Results are presented in Fig. 19, which is a representation of a graphical
output
for ABO blood typing.
EXAMPLE 3
Reverse Typing Assay on Bio-Disc with Sample Preparation Off-Disc
In the example to follow, the reverse blood typing assay is conducted on a bio-
disc comprising: (1 ) gold reflective base disc, treated with photo-
lithography to remove
the gold in specific capture zones, with appropriate chemistry placed over the
capture
zones, (2) 25 um thick channel layer, and (3) gold reflective cover disc,
assembled
into a functional bio-disc.
Whole blood is centrifuged at an appropriate speed and time to result in a
pellet of cells and non-hemolyzed serum or plasma. The serum or plasma is
separately mixed with Type A1 and Type B Reagent Red Blood Cells (Ortho
Clinical
Diagnostics). The mixture of the cells and serum or plasma may take place in
test
tubes or directly on the disc in a mixing chamber. If the mixing occurs in a
test tube,
each mixture is then placed in separate channels of the disc. After a short,
room
temperature incubation (2 to 5 minutes) to allow the serum or plasma to
interact with
the reagent red blood cells, the disc is placed into the drive. The automated
agglutination-detection software developed in-house centrifuges the disc,
causing the
agglutinated and/or non-agglutinated cells to travel over the capture zone and
be non-
specifically captured. Excess cells will be centrifuged to the outer edge of
the flow
channel. The disc is scanned with the standard 780nm laser of the optical
drive using
the bottom detector and the software registers.
The program algorithm determines which reagent red blood cells were
agglutinated and assigns an ABO phenotype, based on reverse typing to the
plasma
64
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WO 03/043403 PCT/US02/36792
or serum sample. The entire process takes about 10 minutes from insertion of
disc
into the drive and receiving the reverse blood typing. The forward and reverse
typings
of an individual should be in agreement, signifying the correct typing of that
individual.
Concluding Summary
While this invention has been described in detail with reference to a certain
preferred embodiments, it should be appreciated that the present invention is
not
limited to those precise embodiments. Rather, in view of the present
disclosure which
describes the current best mode for practicing the invention, many
modifications and
variations would present themselves to those of skill in the art without
departing from
the scope and spirit of this invention. The scope of the invention is,
therefore,
indicated by the following claims rather than by the foregoing description.
All
changes, modifications, and variations coming within the meaning and range of
equivalency of the claims are to be considered within their scope.
Furthermore, those skilled in the art will recognize, or be able to ascertain,
using no more than routine experimentation, many equivalents to the specific
embodiments of the invention described herein. Such equivalents are intended
to be
encompassed by the following claims.
