U.S. patent application number 13/830605 was filed with the patent office on 2013-08-08 for microfluidic device for counting biological particles.
This patent application is currently assigned to The Trustees of Columbia University in the City of New York. The applicant listed for this patent is The Trustees of Columbia University in the City of New York. Invention is credited to Neha AGARWAL, Yuk Kee CHEUNG, Curtis D. CHIN, Samuel K. SIA.
Application Number | 20130203157 13/830605 |
Document ID | / |
Family ID | 40226519 |
Filed Date | 2013-08-08 |
United States Patent
Application |
20130203157 |
Kind Code |
A1 |
CHEUNG; Yuk Kee ; et
al. |
August 8, 2013 |
MICROFLUIDIC DEVICE FOR COUNTING BIOLOGICAL PARTICLES
Abstract
A particle counter for analyzing blood has features which
provide for automatic operation and preferably, also provide for
portable use in a low resource setting. In a preferred embodiment,
preferred embodiment, the device is used to obtain CD4 counts for
AIDS diagnosis.
Inventors: |
CHEUNG; Yuk Kee; (Cambridge,
MA) ; SIA; Samuel K.; (New York, NY) ; CHIN;
Curtis D.; (New York, NY) ; AGARWAL; Neha;
(Los Altos Hills, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
New York; The Trustees of Columbia University in the City
of |
New York |
NY |
US |
|
|
Assignee: |
The Trustees of Columbia University
in the City of New York
New York
NY
|
Family ID: |
40226519 |
Appl. No.: |
13/830605 |
Filed: |
March 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12594176 |
Mar 8, 2010 |
8431090 |
|
|
PCT/US08/68869 |
Jun 30, 2008 |
|
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13830605 |
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|
60947345 |
Jun 29, 2007 |
|
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60947384 |
Jun 29, 2007 |
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Current U.S.
Class: |
435/286.5 ;
435/287.2 |
Current CPC
Class: |
Y10T 436/2575 20150115;
G01N 15/0612 20130101; G01N 15/1484 20130101; G01N 2015/008
20130101; G01N 33/56977 20130101 |
Class at
Publication: |
435/286.5 ;
435/287.2 |
International
Class: |
G01N 33/569 20060101
G01N033/569 |
Claims
1-12. (canceled)
13. A portable blood component analysis device, comprising: a
microfluidic unit with a sample chamber; the sample chamber having
a surface augmentation features with biospecific surface configured
to capture particles from blood; and an analyzer component having a
signal detector and a receiving slot configured to receive the
microfluidic unit and align the sample chamber with the signal
detector.
14. The device of claim 13, wherein the surface augmentation
features include a packed bead bed with glass beads.
15. The device of claim 13, wherein the surface augmentation
features include a packed bead bed with glass beads with a diameter
between 50 and 100 .mu.m and the first longitudinal channel
contains a narrow portion with a minimum dimension of less than the
diameter of the glass beads.
16. The device of claim 13, further comprising a pump, a controller
and a solar power source connected to power the pump and
controller.
17. The device of claim 13, wherein the microfluidic unit includes
a tube containing at least one wash, and at least one air bubble,
the tube being reversibly sealed at both ends and configured to
connect with the second longitudinal channel.
18. The device of claim 13, further comprising a pump, a controller
and a solar power source connected to power the pump and
controller, wherein the microfluidic unit includes a tube
containing at least one wash, and at least one air bubble, the tube
being reversibly sealed at both ends and configured to connect with
the second longitudinal channel and wherein the controller is
configured automatically to pump fluids from the tube through the
sample chamber and through the second longitudinal channel.
19. The device of claim 13, wherein the blood particles include CD4
cells.
20. The device of claim 13, further comprising a battery.
21. The device of claim 13, further comprising a controller and
wherein the signal detector is a light intensity detector and the
controller is configured to derive a cell count from a light
intensity signal without imaging.
22. The device of claim 21, wherein the controller is configured to
derive the cell count from a calibration lookup table correlating
cell count against total light intensity from the sample
chamber.
23. The device of claim 18, further comprising a display configured
to output a cell count.
24. The device of claim 23, further comprising a controller and
wherein the signal detector is a light intensity detector and the
controller is configured to derive a cell count from a light
intensity signal without imaging.
25. The device of claim 24, wherein the controller is configured to
derive the cell count from a calibration lookup table correlating
cell count against total light intensity from the sample
chamber.
26. The device of claim 13, further comprising a pump, a controller
and a solar power source connected to power the pump and
controller, wherein the microfluidic unit includes a tube
containing at least one wash, and an a chemiluminescence activator,
the tube being reversibly sealed at both ends and configured to
connect with the second longitudinal channel and wherein the
controller is configured automatically to pump fluids from the tube
through the sample chamber and through the second longitudinal
channel.
27. The device of claim 18, wherein the pump is controlled to vary
a pumping rate depending on a sample or reagent passing through the
sample chamber.
28. The device claim 13, wherein the analyzer includes a pump
configured to generate a vacuum of at least 10 kPa.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Applications 60/947,384 and 60/947,345, filed 29 Jun. 2007 entitled
"Microfluidic Device for Counting Cells" hereby incorporated by
reference in their entireties herein.
BACKGROUND
[0002] Practical HIV diagnostics are urgently needed in
resource-limited settings. While HIV infection can be diagnosed
using simple, rapid, lateral flow immunoassays, HIV disease staging
and treatment monitoring require accurate counting of a particular
white blood cell subset, the CD4(+) T lymphocyte.
[0003] Current systems for providing this function are expensive,
technically demanding and/or time-consuming. For example, CD4
counts may be obtained by conventional flow cytometry. The method
requires a slow flow rate to capture the cells on the surface of
the channel, followed by a quick wash. This is not easy to
implement in a point-of-care device, where the fluid actuation
technique must be fairly simple. They require a microscope and a
camera (or a person) to count the captured cells. This method is
suitable for a lab environment, not point of care.
SUMMARY
[0004] According to an embodiment, the invention is a portable
microfluidic device for counting CD4+ T-cells. In a method
embodiment, the device is used to count CD4+ T-cells and the
information from the counting is used as a key diagnostic criterion
for determining whether to administer antiretroviral therapy to
HIV-infected patients. In the device embodiment, a disposable
microfluidic cassette contains densely packed beads for capturing
T-cells. A portable, self-powered instrument operatively associated
with the microfluidic cassette reads and displays the result of the
counting. Preferably, the device employs bead-based microfluidics
to enhance surface area for cell capture. For example, in an
embodiment, a "double-dam" design is employed which is compatible
with whole blood. Preferably, a small pump is employed to permit
complete compactness and low power requirement. Also, preferably,
the device uses a portable detection device based on an
absorbance/chemiluminescence reader. In a particularly preferred
embodiment, the detector is powered by a solar batter, which can,
for example facilitate its use in point-of-care settings in the
developed countries, as well as developing countries.
[0005] In a particular variation, the device uses cell affinity
chromatography operated under differential shear flow to
specifically isolate CD4(+) T lymphocytes. In an embodiment, the
device may be effective with a direct processing of 10 microliters
of unprocessed, unlabeled whole blood. CD4 counts are, in this
embodiment, obtained under an optical microscope in a rapid, simple
and label-free fashion. For example, the embodiments are effective
for a range of absolute CD4 counts (R(2)=0.93) over which the
embodiments are operable. This CD4 counting microdevice can be used
for simple, rapid and affordable CD4 counting in point-ofcare and
resource-limited settings.
[0006] In a preferred configuration, packed beads are provided in a
microchannel to enhance capture surface area which increases the
cell-capture efficiency to enhance the final signal, and allows
constant flow, permitting the use of a small pump, preferably
peristaltic and preferably powered by a battery. Preferably,
capture and detection are done in the same location. Optics are
employed that automatically reads the signal either by absorbance
or chemiluminescence or some other suitable technique. The optical
components are preferably portable and as such, are, in the
preferred embodiment, powered by battery including the detection
portion, the pumping portion, and the microfluidic channel chip.
Preferably, rechargeable batteries are used.
[0007] A portable blood component analysis device has a
microfluidic unit with a sample chamber defined in a first
longitudinal channel having a first major axis and a second
longitudinal channel, having a second major axis, in communication
with the first longitudinal channel with the second major axis
crossing the first major axis; the sample chamber having a surface
augmentation features with biospecific surface configured to
capture particles from blood; an analyzer component having a signal
detector and a receiving slot configured to receive the
microfluidic unit and align the sample chamber with the signal
detector. Preferably, the first and second major are substantially
perpendicular. Preferably, the analyzer contains a pump configured
to generate a vacuum of at least 10 kPa. Preferably, the surface
augmentation features include a packed bead bed with glass beads.
Preferably, the surface augmentation features include a packed bead
bed with glass beads with a diameter between 50 and 100 .mu.m and
the first longitudinal channel contains a narrow portion with a
minimum dimension of less than the diameter of the glass beads.
Preferably, the device further includes a pump, a controller and a
solar power source connected to power the pump and controller.
Preferably, the microfluidic unit includes a tube containing at
least one, at least one wash, and at least one air bubble, the tube
being reversibly sealed at both ends and configured to connect with
the second longitudinal channel. Preferably, the device further
includes a pump, a controller and a solar power source connected to
power the pump and controller, wherein the microfluidic unit
includes a tube containing at least one, at least one wash, and at
least one air bubble, the tube being reversibly sealed at both ends
and configured to connect with the second longitudinal channel and
wherein the controller is configured automatically to pump fluids
from the tube through the sample chamber and through the second
longitudinal channel. In any of these devices, the blood particles
detected may include CD4 cells. Preferably, a battery is included.
Preferably, a controller receives a signal from the signal detector
and the signal is a light intensity, the controller being
configured to derive a cell count from a light intensity signal
without imaging. Preferably, the controller is configured to derive
the cell count from a calibration lookup table correlating cell
count against total light intensity from the sample chamber.
[0008] Another portable blood component analysis device has a
microfluidic unit with a sample chamber; the sample chamber having
a surface augmentation features with biospecific surface configured
to capture particles from blood. an analyzer component having a
signal detector and a receiving slot configured to receive the
microfluidic unit and align the sample chamber with the signal
detector; Preferably, the surface augmentation features include a
packed bead bed with glass beads. The device claim 13, wherein the
analyzer contains a pump configured to generate a vacuum of at
least 10 kPa; Preferably, the surface augmentation features include
a packed bead bed with glass beads with a diameter between 50 and
100 .mu.m and the first longitudinal channel contains a narrow
portion with a minimum dimension of less than the diameter of the
glass beads. Preferably, the device includes a controller and a
solar power source connected to power the pump and controller.
Preferably, the microfluidic unit includes a tube containing at
least one, at least one wash, and at least one air bubble, the tube
being reversibly sealed at both ends and configured to connect with
the second longitudinal channel. Preferably, the device has a pump,
a controller and a solar power source connected to power the pump
and controller, wherein the microfluidic unit includes a tube
containing at least one, at least one wash, and at least one air
bubble, the tube being reversibly sealed at both ends and
configured to connect with the second longitudinal channel and
wherein the controller is configured automatically to pump fluids
from the tube through the sample chamber and through the second
longitudinal channel. Preferably, the blood particles include CD4
cells. Preferably, the device has a battery powered by solar power
source. Preferably, the device has a controller and the signal
detector is a light intensity detector and the controller is
configured to derive a cell count from a light intensity signal
without imaging. Preferably, the controller is configured to derive
the cell count from a calibration lookup table correlating cell
count against total light intensity from the sample chamber.
Preferably, the device has a display configured to output a cell
count. Preferably, the device has a controller and the signal
detector is a light intensity detector and the controller is
configured to derive a cell count from a light intensity signal
without imaging. The device of claim 23, wherein the controller is
configured to derive the cell count from a calibration lookup table
correlating cell count against total light intensity from the
sample chamber. Preferably, the device includes a pump, a
controller and a solar power source connected to power the pump and
controller, wherein the microfluidic unit includes a tube
containing at least one, at least one wash, and an a
chemiluminscence activator, the tube being reversibly sealed at
both ends and configured to connect with the second longitudinal
channel and wherein the controller is configured automatically to
pump fluids from the tube through the sample chamber and through
the second longitudinal channel. Preferably, the pump is controlled
to vary a pumping rate depending on a sample or reagent passing
through the sample chamber.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 shows a first embodiment of a sample analyzer.
[0010] FIGS. 2A and 2B show a second embodiment of a sample
analyzer.
[0011] FIG. 3 shows a multiple-chamber sample analyzer showing
various features.
[0012] FIGS. 4A, 4B, and 4C illustrate embodiments of portable
sample analyzer machines.
DETAILED DESCRIPTION OF EMBODIMENTS
[0013] Referring to FIG. 1, a T-shaped microfluidic device 100 has
a sample channel 100S for sample fluid and reagents and a loading
channel 100L for loading particles that providing surface
augmentation, such as glass or polystyrene beads 106. The particles
preferably carry immobilized biospecific molecules or other
reactive substances on their surfaces. An inlet channel 101 and an
outlet channel 102 are used to convey a sample fluid, such as
blood, and reagents and washes through the sample channel 100S. The
loading channel 100L is used to fill a chamber 104 with the surface
augmentation particles to form a packed bed within the chamber 104.
The beads 106 are trapped in a cage made up of microposts 108
defining sample inlet, sample outlet fences 110, 112, and 114. The
beads may be suspended in a fluid and conveyed through a loading
port 107 along with the fluid suspending them into the chamber 104
while permitting the fluid to drain through gaps between the posts
108. The beads may be loaded in by flowing them in a suspension by
pumping a fluid into the bed 104 area and drawing excess carrying
fluid out through a fence 114 trapping the beads 106 in the bed 105
and packing them with a repeatable density.
[0014] Preferably, the gap between the posts is smaller than the
breads. The Posts 108 can be many times the size of the beads. The
beads do not have to be a constant size, but preferably they are a
consistent size to help ensure a packing arrangement with a large
and predictable void volume. If desired, a step may be added, for
example, to coat the bead particles with a stabilizing or
protecting material.
[0015] Although not shown in the drawing, the chamber 104 is
enclosed at the top and bottom. The entire structure can be made
using lithographic techniques that are well known. Once packed with
the surface augmenting particles, loading channel 100L can be
permanently sealed at both ends and the sample channel 1005
temporarily sealed for storage and/or shipping. The device 100 may
be combined with multiple such devices 100 on a single piece of
substrate, each with a bed having a different immobilized
biospecific substances to permit detection of different sample
fluid components. In use, the sample channel 100L is unsealed such
that fluid can enter and leave chamber 104 through the inlet and
outlet channels 101 and 102. The drawing does not show the beads
106 in place in the bead bed 104.
[0016] Referring to FIGS. 2A and 2B, an array of sample channels
231, 232, 233, and 234, each having a sample chamber 218, is formed
in a substrate 201. The sample chambers 218 are functionally
similar to the chamber 104 of FIG. 1, except that in the present
embodiment, narrow channels 216 are used to retain the surface
augmentation particles 106, for example, beads. The beads are
loaded into loading ports 202 (as indicated by arrow 237--only one
port 202 is labeled to avoid crowding the drawing, but the others
are similarly configured). The beads are held back by the narrow
channels 216 and the fluid carrying the beads exits as shown by the
arrow 239 out of an exit port 204. Using these structures, beads
with different immobilized substances can be loaded in each sample
chamber 218. Once the sample chambers 218 are packed, the ports 202
and 204 are sealed and the device 200 can be used or stored and/or
shipped. In other respects, the device 200 is similar to that
described with reference to FIG. 1.
[0017] In use, the sample, washes, and reagents (collectively
indicated by arrow 235) are loaded through sample inlet ports 212
and spent fluids recovered or disposed of through sample outlet
port 206 (arrow 237). As described below, sample channels 210
convey a fluid train (the sample, washes, and reagents) serially
through each of the sample chambers. Preferably, the fluid train is
drawn through the device 200 using a vacuum pump regulated to
maintain a pressure that provides desired shear rate which has been
determined to remove particles that do not bind with the beads
(more specifically, the active surface of the bead particles) and
permit those particles (such as cells) that do bind to remain in
the sample chambers 218. In certain embodiments, it will preferably
be determined the range of shear rates that provide the desired
discrimination behavior.
[0018] FIG. 2B illustrate, as viewed from the top, a single sample
chamber 218 with beads 240 packed within. The sample channel 210
permits the train of sample fluid and reagents to pass through.
Preferably, the sample line has a size that prevents the migration
of substantial quantities of beads 240 from the sample chamber 218.
In an embodiment, the sample channel width is selected to be
slightly smaller than the coated beads.
[0019] As will be described below, sample fluid particles in sample
fluid, such as CD4 cells (particles) in blood (sample fluid) flow
into the sample chamber followed by a wash, followed by a fluid
carrying free signal molecules, followed by a wash, followed by a
fluid carrying a free signal amplifier molecule. In each case, a
different shear rate may be desirable to ensure that only the
reactive particles or molecules are left behind and such that the
shear rate is not so high as to interfere with the ability of
particles to attach ultimately to the beads or substances thereon.
The shear rate may vary depending on the component of the assay,
for example, when a wash is applied versus when a reagent is
applied. Shear rate may vary for different reagents as well.
Preferably, an automated device, as described below, is provided
with a controller to vary the total vacuum to vary the shear rate
accordingly. This may be synchronized to the stage of the assay by
detecting a property of the fluid train (e.g. detecting air bubbles
that isolate the fluids from each other), by time after start of
the vacuum pump, by detecting momentary changes in the vacuum
pressure caused by passage of different fluids, such as air, into
the beds, or other means.
[0020] Referring to FIG. 3, beads 240 configured for capturing the
target cells (such as by anti-target cell antibody) are packed in a
bed 218 in an analyzer device 300. The device has tubing 312 with
connectors 356 that connect to sample fluid train 351 and a vacuum
pump 306. The sample fluid train may be held in a long tube 307
which is sealed with rupturable seals 350. The rupturable seals may
be configured to rupture upon application of a vacuum or by being
pierced automatically by a sharp edge (not shown) when installed in
a portable device. After the sample fluid train passes through the
device 300, a signal detector 302 may detect light caused by a
reaction, for example, chemiluminescence or fluorescence, In the
latter case, a light source, such as photodiode 304 may be
provided. In a preferred embodiment, the number of cells captured
in the sample chamber is the quantity of interest and preferably
the system is configured such that the total amount of light
captured by the detector indicates the number of cells according to
a predetermined calibration curve stored in a portable controller
and the cell count or other indication displayed automatically by
the device.
[0021] The train of fluid 310 including sample fluid 320, such as
whole blood conveyed through an inlet channel 312 to the bed 218.
The sample fluid train may include further fluids, each separated
from each other, as appropriate in view of their mutual activity,
by air or gas bubbles 340. The fluid train may also include washes
322, 328, fluids carrying intermediate materials such as antibodies
that bind signal molecules, fluids carrying signal molecules 324,
developers and, fluids carrying signal amplifiers 326, etc. For
another example, the sample fluid train may include substance that
causes chemoluminescence.
[0022] In a preferred embodiment, the target that reacts with the
reactive substance on the beads is lymphocytes (for example CD4
cells) carried in the sample fluid, blood. In such embodiment, is
followed by a wash and then a target cell antibody (the primary
antibody). In the principal embodiment, this is a CD4 cell primary
antibody; i.e., an antibody that recognizes a CD4 cell. Following
that, a quantity of a detectable (labeled) secondary antibody that
recognizes the CD4 cell antibody is conveyed through the inlet
channel and binds or associates with the primary antibody. A
developer may be conveyed as well to enhance or permit
detection.
[0023] As mentioned, to detect the quantity of target cells
retained in the sample chambers, the device preferably measures the
strength of an optical signal. Examples included fluorescence,
label (e.g. silver) absorbance, and chemiluminescence. Techniques
which are known in the prior art, may be employed. A small
electronic (charge-coupled device; "CCD") camera, microscope, or
other suitable photodetector device can be employed with suitable
optics and filtering to provide an indicator signal. The total
magnitude of the light signal may be compared to a standard to
quantify the count of cells.
[0024] In an exemplary embodiment, the embodiments employ
dimensions that are determined to permit the close packing in a
single layer while permitting sufficient flow of sample fluid and
reagents without clogging. The dimensions of the embodiment of FIG.
1 may include 90 .mu.m-diameter microposts 108 spaced apart a
distance smaller than the beads 108, for example, spaced 25 .mu.m
apart to trap them. The beads are 40 .mu.m-diameter and the depth
(dimension parallel to the axes of the microposts 108) of the
channel defined by the bed 104 is 75 .mu.m-tall channels. In an
exemplary embodiment, the bed 104 is 2 mm by 2 mm. The void
fraction of the packed bed 104 may be chosen such that cells clear
the column after a few washes.
[0025] Referring to FIG. 4A, a schematic of a portable analyzer 400
has a power source 404, for example, a solar cell or hand
generator. A microprocessor based microcontroller (XTL) 402
controls the unit and drives a pump, (optionally, if used) a light
source (e.g., laser diode) or other exciter 412, photodetector 410,
and a display 408, preferably an LCD. Inputs from a user may be
provided by suitable actuators such as membrane switches to allow a
user to operate the device. Switches, 418 and 420 may be limited to
power on and run functions to make the unit fully automated and
simple to operate. A housing 422 is preferably provided to form a
unitary portable device.
[0026] Referring to FIG. 4B, a more detailed embodiment of a
portable analyzer 450 has a vacuum pump 462 to generate a vacuum
for drawing sample and reagents through a microfluidic chip 494 as
discussed above. A pressure sensor 471 detects a pressure signal
via a pressure chamber 464 which smooths out pressure pulses,
indicating the vacuum output of the vacuum pump. Another pressure
sensor 470 detects a pressure signal via a pressure chamber 468
which also smooths out pressure pulses, the latter signal
indicating the regulated vacuum pressure determined by a pressure
regulator 466. The pressure signals are applied to a
microcontroller 488 as are signals from user control actuators 472,
and photodetector signals from one or more photodetectors 476
amplified by an amplifier 480. The signal from the photodetectors
476 may are preferably sampled and converted to digital data and
processed for analysis to permit results to be displayed on a
display 482. Further output devices may include an indicator
annunciator 484 and/or an indicator lamp 478. The latter may be
used, for example, to indicate the completion of an analysis and
the status of the device 450, respectively, for example.
[0027] Referring to FIG. 4C, in a preferred embodiment, a
microfluidic chip 604 with reagent cache 602 (tube filled with
reagent and having a sample port 603) is configured to be received
in a recess 606 of an analyzer 608 to form a configuration as
described with reference to FIG. 4A or 4B. In the present
embodiment, a swing-out front panel has a solar charger 614.
Control buttons such membrane switches (not shown) may be provided
on the housing. A circuit board (not shown) carrying an LCD display
615 may be carried on the front panel. The microfluidic channel
"chip" 604 (and fluid train cache 602) may be provided in the form
of a replaceable component which is used once for each measurement
and discarded. In a following layer a circuit board carries a
microcontroller and photodetector (or camera or other sensor)
positioned to detect light from the bead bed. Batteries and a pump
may be carried in a lower chassis. The entire structure shown can
be packaged in a hinged device which may be opened to insert the
microfluidic chip while permitting access for the drawing of a
sample fluid (and any detection or development agents
required).
[0028] In an exemplary embodiment, the laser diode may, for
example, have a wavelength of 654 nm. The device may have an
alignment slot, which is exposed when opened, to permit the proper
location of the microfluidic chip. It may also have a
Polydimethylsiloxane window to protect the detector and/or the
light source. In an embodiment, the photodetector records changes
in absorbance, for example due to opacity of a developing silver
film. In another embodiment, the photodetector detects light
emitted by captured sample material and labels, e.g., by
fluorescence. Preferably, results are displayed digitally to
minimize subjective interpretation by the user.
[0029] To confirm the above configuration, a passive adsorption of
antibodies onto beads was tested. The capture of 3T3 fibroblasts on
the beads (coated with human fibronectin), and CD4 T-cells (using
anti-CD4 antibodies) has been demonstrated by experiment. An
ability to capture both fibroblasts and lymphocytes with this
method (FIG. 2A), has been confirmed. The use of covalent chemistry
has been investigated, using carboxylated beads and the
carbodiimide coupling agent EDC. Beads coated with anti-CD4
successfully captured T-cells from whole blood of AIDS patients, by
DIC (FIG. 2B) and fluorescence microscopy (FIG. 2C). Among
commercially available antibodies, it was found that only a small
number have strong affinity for CD4 and can be easily coated onto
surfaces. Using this set of antibodies, we will finish a
signal-to-background optimization by July 31 as scheduled.
[0030] To prepare glass beads, the following methodology was
experimentally confirmed as suitable for use in the above
embodiments. First, beads having a diameter of about 50 .mu.m were
subjected to 2N nitric acid wash for 1 hour (to clean the glass
beads). Next, the beads were silanized with 10% APTES (an
aminosilane) overnight at room temperature. The beads were washed
with toluene and acetone, and dried in an oven at 60C for 2 hr.
Then the beads were reacted with 2.5% glutaraldehyde (to crosslink
terminal--NH2 on APTES with primary amines on proteins added later)
for 1 hour at room temperature and subsequently washed with
distilled water. The next step was to couple 10 .mu.g/ml of protein
G onto silanized glass surface (to orient anti-CD4+ antibody with
F(ab) towards mobile phase, and to act as additional spacer). The
next step was to wash 3 times with phosphate buffer saline solution
and couple 10 .mu.g/ml of mouse-anti-human CD4+ antibody onto
protein-G coated glass surface. Specific binding of fluorescent
antibodies was demonstrated, using bovine serum albumin (BSA) as a
negative control to block binding of fluorescent secondary antibody
markers (AF-488 goat anti-mouse antibodies).
[0031] It has been experimentally determined that beads of a
certain minimum size are preferred to reduce clogging by cell
particles. In balancing the size against the reduction in surface,
the preferred size range has been determined to be in the range of
50-100 and preferably about 75-80 .mu.m diameter. An exemplary
shear rate determined to provide reliable discrimination is 10-20
dyn/cm.sup.2.
* * * * *