U.S. patent application number 15/393596 was filed with the patent office on 2017-04-13 for microfluidic device for real-time clinical monitoring and quantitative assessment of whole blood coagulation.
The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to Donald E. Ingber, Abhishek Jain, Daniel C. Leslie, Mike Super, Anna Waterhouse.
Application Number | 20170100714 15/393596 |
Document ID | / |
Family ID | 53494200 |
Filed Date | 2017-04-13 |
United States Patent
Application |
20170100714 |
Kind Code |
A1 |
Jain; Abhishek ; et
al. |
April 13, 2017 |
Microfluidic Device For Real-Time Clinical Monitoring And
Quantitative Assessment Of Whole Blood Coagulation
Abstract
A microfluidic coagulation assessment device includes a
plurality of microchannels, with a blood sample driven through the
microchannels at a substantially constant flow rate. A controller
is configured to, in combination with a timer and a pressure
sensing device, determine a first pressure value (or flow value) at
an initiation of flow, a first time (T.sub.pg) at which a second
pressure value is about twice the determined first pressure value,
and a second time (T.sub.pf) at which a third pressure value is
about (1+e) times the determined first pressure value and establish
a subject coagulation model predictive of channel occlusion
therefrom.
Inventors: |
Jain; Abhishek; (Roslindale,
MA) ; Waterhouse; Anna; (Brookline, MA) ;
Super; Mike; (Lexington, MA) ; Ingber; Donald E.;
(Boston, MA) ; Leslie; Daniel C.; (Brookline,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Family ID: |
53494200 |
Appl. No.: |
15/393596 |
Filed: |
December 29, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15028667 |
Apr 11, 2016 |
9562914 |
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PCT/US14/60956 |
Oct 16, 2014 |
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15393596 |
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61891732 |
Oct 16, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/50273 20130101;
B01L 2200/146 20130101; B01L 3/502746 20130101; B01L 2400/0478
20130101; B01L 2300/0883 20130101; B01L 2300/08 20130101; G16B
40/00 20190201; G01N 33/4905 20130101; B01L 2400/084 20130101; B01L
2300/0627 20130101; G01N 33/86 20130101; B01L 2300/0816 20130101;
B01L 2300/0861 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; G01N 33/49 20060101 G01N033/49; G06F 19/24 20060101
G06F019/24; G01N 33/86 20060101 G01N033/86 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] Some aspects of the present disclosure were made with
government support, under Grant No. N66001-11-1-4180 and
HR0011-13-C-0025 awarded by the Defense Advanced Research Projects
Agency (DARPA), and the government shares rights to such aspects of
the present disclosure.
Claims
1. A microfluidic coagulation device, comprising: at least one
substrate defining a plurality of microchannels; a first port at a
first end portion of the substrate, the first port connecting to
first ends of the plurality of microchannels; a second port at a
second end portion of the substrate, the second port connecting to
second ends of the plurality of microchannels; a first sensing
device configured to determine a pressure value in, or relating to,
a pressure across the plurality of microchannels; a timer; and a
controller configured to determine, in combination with the first
sensing device and the timer, a first pressure value at an
initiation of flow, a first time at which a second pressure value
is determined to be about twice the determined first pressure
value, and a second time at which a third pressure value is about
(1+e) times the determined first pressure value, and further
configured to establish a subject coagulation model predictive of
channel occlusion in accord with the relation .DELTA. P ( t )
.DELTA. P ( 0 ) = 1 + t - T pg T pf - T pg ##EQU00021## wherein
T.sub.pf is the second time and T.sub.pg is the first time, and
wherein a differential pressure or flow rate/shear applied across
the first port drives a blood sample across the plurality of
microchannels at a substantially constant flow rate.
2-86. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/891,732, filed Oct. 16, 2013, the
contents of which are incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates generally to microfluidic
diagnostic devices, systems and methods and, more particularly, to
a microfluidic diagnostic devices, systems and methods for
real-time assessment of whole blood coagulation.
BACKGROUND OF THE INVENTION
[0004] Prevention of thrombosis with anticoagulants, such as
heparin or coumadin, is critical for treatment of many diseases and
conditions (e.g., atrial fibrillation, sepsis, trauma, prosthetic
heart valves, various coagulapathies or other bleeding disorders)
as well as for many life-saving procedures, including dialysis,
hemofiltration, extracorporeal oxygenation (ECMO), angioplasty,
intravenous fluid delivery, apheresis and collection of blood
samples for analysis or culture. As thrombosis can result from
activation of platelets as well as the coagulation cascade,
anticoagulation therapy is typically supplemented by anti-platelet
therapy. It would therefore be extremely helpful to be able to
monitor global anticoagulation in real-time in the clinic because
coagulation responses to variations in anticoagulant levels and
platelet numbers can vary significantly among patients and in the
same patient at different times. However, it is currently difficult
to quickly and quantitatively ascertain the degree and efficiency
of whole blood anti-coagulation and anti-platelet therapies, and
there is no reliable method to do it rapidly at the bedside, having
the possibility of using native blood.
[0005] Patients who have bleeding or clotting problems are now
routinely monitored using Prothrombin Time (PT) and Activated
Clotting Time (ACT)/Activated Partial Clotting Time (APTT) tests,
which provide semi-quantitative measures of the extrinsic or
intrinsic coagulation pathways respectively. However, the results
produced by these assays can vary considerably depending on sample
preparation, anticoagulation tubes, addition of activators,
equipment, and user expertise. As a result, measurements of the
same sample carried out at different sites or on different days
often produce different results. The specificity and sensitivity of
these tests are also poor and often result in false positive or
negative cases in the clinic. Moreover, PT, ACT and APTT assays do
not provide information on platelet function and therefore, do not
serve as global coagulation tests. Because of the limitations of
conventional blood clotting time tests, new point-of-care
monitoring systems, such as thromboelastography (TEG) and rotation
thromboelastometry (ROTEM) devices, have started to be integrated
into clinical laboratories. These devices are able to provide
greater information about hemostasis because they measure the
cumulative contribution of plasma, platelets, leukocytes and red
blood cells to the clotting response. These tests, however, measure
clotting characteristics under static conditions (no flow) and
hence, they are limited in their clinical utility with respect to
platelet and endothelial cell functions, which are highly sensitive
to physical forces, including pressure and flow. For example, fluid
shear stresses and gradients of shear stresses in blood have a
major impact on platelet activation and thrombosis and thus,
coagulation monitors that do not incorporate fluid dynamics fail to
accurately assess blood coagulation physiology as it occurs in the
vasculature of a living patient.
SUMMARY OF THE INVENTION
[0006] A coagulation monitoring technology that incorporates
relevant hemodynamic mechanical cues (shear stress and gradients)
and that can be carried out with minimal (or none) sample
preparation or operator training, in vitro or ex vivo, and
integrated with extracorporeal blood perfusion systems (e.g.,
dialysis, hemofiltration, ECMO) would greatly enhance hemostasis
assessment and patient or subject care (e.g., human
patient/subject, animal patient/subject) capabilities in the clinic
or laboratory. The microfluidic devices, systems and methods
disclosed herein present an opportunity to fill this clinical unmet
need by developing physiologically relevant bedside or lab bench
tests that can help to both unravel the dynamics of thrombosis and
aid in quantitative analysis of clot formation under both
physiological and pathological fluid shear stress conditions, with
the possibility of attaching the device directly to the patient
blood vessel.
[0007] By way of example, and as discussed in more detail below,
one embodiment of at least some of the present concepts comprises a
microfluidic device comprising polydimethylsiloxane (PDMS) in which
a network of rectangular microchannels (75.times.200 .mu.m) are
defined, these microchannels being approximately equivalent in size
to 125 .mu.m diameter living arterioles (FIG. 1). This microfluidic
device and system is suitable for clinical or point-of-care use
(e.g., bedside-capable) and is configured to measure thrombotic
potential and platelet aggregation of whole blood (e.g., human
blood, animal blood) in real-time.
[0008] In accord with the present concepts, full occlusion of
microchannels in the device due to clot formation can be, for
example, dynamically measured while independently controlling the
concentration of anticoagulant (e.g., unfractionated heparin) or
applied wall shear stress and gradient of shear stress.
[0009] Contrary to current static coagulation assessment devices,
which ignore that thrombosis of blood vessels in vivo depends on
the way blood flow in the circulation is maintained (e.g., the
human heart pumps blood such that a relatively constant flow rate
is maintained in the arterial circulation, while nearly constant
pressure is sustained in the venous circulation), at least some
aspects of the disclosed coagulation monitoring microfluidic device
are configured to deliver blood in a substantially constant flow
mode of operation and/or substantially constant pressure mode of
operation using either a syringe pump or a constant-pressure pump,
respectively. The parameters of substantially constant flow or
substantially constant pressure also can be varied independently,
if desired.
[0010] In accord with at least some aspects of the present
concepts, clotting within the microfluidic channel is characterized
by recording the rise in pressure (when flow is substantially
constant in a constant flow mode of operation) or drop in flow rate
(when pressure is substantially constant in a constant pressure
mode of operation) and, using experimentally-validated
phenomenological mathematical models described herein, develop one
or more patient-specific or subject-specific predictive models for
the temporal dynamics of whole blood clotting. The characteristic
time constants of these respective models represent the clotting
times of blood under shear flow. As these time constants are
patient-specific or subject-specific, they can be determined by
clinicians as a routine diagnostic test to quantitate, monitor, and
track thrombogenicity, platelet function, bleeding disorders and
anti-coagulation therapy under physiologically relevant
conditions.
[0011] Current coagulation monitoring instruments have high
variability, and often report unreliable and non-physiological
clotting times. The present inventors have determined that the
accuracy of these current coagulation monitoring instruments is low
because they fail to measure contributions of blood rheology and
hydrodynamic shear stresses and their gradients to hemostasis and
thrombosis, which can vary from patient-to-patient and even
day-to-day. Accordingly, the present concepts concern microfluidic
devices, systems and methods that incorporate relevant flow
hemodynamics and provide a quantitative measure of clotting
activity that can significantly improve clinical assessment of
blood coagulation, and which can be advantageously integrated with
other systems, such as an extracorporeal blood perfusion
devices.
[0012] In accord with one aspect of the present concepts, a
microfluidic coagulation device, comprises at least one defining a
plurality of microchannels, a first port at a first end portion of
the substrate, the first port connecting to inlet ends of the
plurality of microchannels, and a second port at a second end
portion of the substrate, the second port connecting to outlet ends
of the plurality of microchannels. An instrument that causes
differential pressure or flow rate/shear across the first port is
provided to drive a blood sample across the plurality of
microchannels at a substantially constant flow rate. A first
sensing device is configured to determine a pressure value in, or
relating to, a pressure across the plurality of microchannels and a
timer is provided to measure time. Further, a controller, which may
comprise one or more processors which may be local and/or remote,
is configured to determine, in combination with the first sensing
device and the timer, a first pressure value at an initiation of
flow, a first time at which a second pressure value is determined
to be about twice the determined first pressure value, and a second
time at which a third pressure value is about (1+e) times the
determined first pressure value, and further configured to
establish a patient coagulation model predictive of channel
occlusion in accord with the relation
.DELTA. P ( t ) .DELTA. P ( 0 ) = 1 + t - T pg T pf - T pg
##EQU00001##
wherein T.sub.pf is the second time and T.sub.pg is the first
time.
[0013] In another aspect of the present concepts, a microfluidic
coagulation device, comprises at least one substrate defining a
plurality of microchannels, a first port at a first end portion of
the substrate, the first port connecting to inlet ends of the
plurality of microchannels, and a second port at a second end
portion of the substrate, the second port connecting to outlet
endsof the plurality of microchannels. An instrument to apply a
differential pressure across the first port is attached to the
first port to apply a differential pressure across the first port
to drive a blood sample across the plurality of microchannels. A
first sensing device is configured to determine a flow rate in, or
relating to, the plurality of microchannels and a timer is provided
to measure time. A controller, which may comprise one or more
processors which may be local and/or remote, is configured to
determine, in combination with the first sensing device and the
timer, (i) a first flow rate value at a first time corresponding to
an initiation of flow, (ii) a second time at which a second flow
rate value is determined to be about half the determined first flow
rate value, (iii) a third time at which a third flow rate value is
determined to be about (1+e) times lesser than the determined first
flow rate value, and (iv) a patient coagulation model predictive of
channel occlusion governed by the relation
.DELTA. P ( t ) .DELTA. P ( 0 ) = 1 + t - T pg T pf - T pg
##EQU00002##
wherein T.sub.qf is the third time and T.sub.qg is the second
time.
[0014] In yet another aspect of the present concepts, a method of
assessing coagulation of a subject's blood comprises the acts of
driving a blood sample from the subject at a substantially constant
flow rate through a plurality of microchannels formed in a
microfluidic device substrate and measuring a pressure, or a
variable correlated with pressure, in at least one of the plurality
of microchannels while the blood sample is moved through the
plurality of microchannels at the substantially constant flow rate.
The method also includes the acts of determining a first pressure
value at an initiation of flow, determining a first time at which a
second pressure value is determined to be about twice the
determined first pressure value, and determining a second time at
which a third pressure value is determined to be about (1+e) times
the determined first pressure value. The method further includes
the act of establishing a subject-specific coagulation model
predictive of channel occlusion for the subject using the first
time and the second time in the relation
.DELTA. P ( t ) .DELTA. P ( 0 ) = 1 + t - T pg T pf - T pg
##EQU00003##
wherein T.sub.pf is the second time and T.sub.pg is the first time.
The method further includes the act of recording, on a physical
storage medium, the established subject-specific coagulation
model.
[0015] In yet another aspect of the present concepts, a method of
assessing coagulation of a subject's blood comprises the acts of
driving a subject's blood sample at a substantially constant
pressure through a plurality of microchannels formed in a
microfluidic device substrate and measuring a flow rate, or a
variable correlated with flow rate, in at least one of the
plurality of microchannels while the blood sample is moved through
the plurality of microchannels at the substantially constant
pressure. The method further includes the acts of determining a
first flow rate value at an initiation of flow, determining a first
time at which a second flow rate value is determined to be about
half the determined first flow rate value, determining a second
time at which a third flow rate value is determined to be about
(1+e) times lesser than the determined first flow rate value, and
establishing a subject-specific coagulation model predictive of
channel occlusion for the subject using the first time and the
second time in the relation
Q ( t ) Q ( 0 ) = 1 1 + t - T qg T qf - T qg ##EQU00004##
wherein T.sub.qf is the second time and T.sub.qg is the first time.
The method also includes the act of recording, on a physical
storage medium, the established subject-specific coagulation
model.
[0016] In yet another aspect of the present concepts, a method of
assessing coagulation of a subject's blood comprises the acts of
driving a first blood sample for the subject at a substantially
constant flow rate through a first plurality of microchannels
formed in a first microfluidic device substrate and measuring a
pressure, or a variable correlated with pressure, in at least one
of the first plurality of microchannels while the first blood
sample is moved through the first plurality of microchannels at the
substantially constant flow rate. The method also includes the acts
of determining a first pressure value at an initiation of flow,
determining a first time at which a second pressure value is
determined to be about twice the determined first pressure value,
and determining a second time at which a third pressure value is
determined to be about (1+e) times the determined first pressure
value. The method further includes the act of establishing a first
subject coagulation model predictive of channel occlusion using the
first time and the second time in the relation:
.DELTA. P ( t ) .DELTA. P ( 0 ) = 1 + t - T pg T pf - T pg
##EQU00005##
wherein T.sub.pf is the second time and T.sub.pg is the first time.
The method further includes recording, on a physical storage
medium, the established first subject-specific coagulation model.
Still further, the method includes driving a second blood sample
for the subject at a substantially constant pressure through a
plurality of second microchannel formed in the first microfluidic
device substrate or in a second microfluidic device substrate and
measuring a flow rate, or a variable correlated with flow rate, in
at least one of the plurality of second microchannels while the
blood sample is moved through the plurality of second microchannels
at the substantially constant pressure. The method further includes
determining a first flow rate value at an initiation of flow,
determining a first time at which a second flow rate value is
determined to be about half the determined first flow rate value,
determining a second time at which a third flow rate value is
determined to be about (1+e) times lesser than the determined first
flow rate value, and establishing a second subject-specific
coagulation model predictive of channel occlusion using the first
time and the second time in the relation
Q ( t ) Q ( 0 ) = 1 1 + t - T qg T qf - T qg ##EQU00006##
wherein T.sub.qf is the second time and T.sub.qg is the first time.
The method further includes recording, on the physical storage
medium, the established second subject-specific coagulation
model.
[0017] In yet another aspect of the present concepts, a method of
assessing coagulation of a subject's blood, comprises the acts of
driving a blood sample at a substantially constant flow rate
through a plurality of microchannels formed in a microfluidic
device substrate and measuring a pressure, or a variable correlated
with pressure, in at least one of the plurality of microchannels
while the blood sample is moved through the plurality of
microchannels at the substantially constant flow rate. The method
further includes the acts of determining a first pressure value at
an initiation of flow, determining a first time at which a second
pressure value is determined to be about twice the determined first
pressure value, and determining a second time at which a third
pressure value is determined to be about (1+e) times the determined
first pressure value. The method also includes the act of
establishing a subject coagulation model predictive of channel
occlusion and recording, on a physical storage medium, clotting
times, utilizing the relation
( T pg , T pf ) = A ( T pg , T pf ) B ( T pg , T pf ) C uh - C ( T
pg , T pf ) .gamma. ##EQU00007##
wherein A, B and C are subject specific variables relating to blood
properties empirically determined by curve fitting the following
relation
.DELTA. P ( t ) .DELTA. P ( 0 ) = 1 + t - T pg T pf - T pg
##EQU00008##
and wherein T.sub.pf is the second time and T.sub.pg is the first
time.
[0018] In yet another aspect of the present concepts, a method of
assessing coagulation of a subject's blood comprises the acts of
driving a blood sample at a substantially constant pressure through
a plurality of microchannels formed in a microfluidic device
substrate and measuring a flow rate, or a variable correlated with
flow rate, in at least one of the plurality of microchannels while
the blood sample is moved through the plurality of microchannels at
the substantially constant pressure. The method also includes the
acts of determining a first flow rate value at an initiation of
flow, determining a first time at which a second flow rate value is
determined to be about half the determined first flow rate value,
and determining a second time at which a third flow rate value is
determined to be about (1+e) times lesser than the determined first
flow rate value. The method also includes the acts of recording, on
a physical storage medium, clotting times utilizing the
relation
( T qg , T qf ) = A ( T qg , T qf ) .gamma. .omega. B ( T qg , T qf
) C uh - C ( T qg , T qf ) .gamma. ##EQU00009##
wherein A, B C and .omega. are subject specific variables relating
to blood properties empirically determined by curve fitting the
following relation
Q ( t ) Q ( 0 ) = 1 1 + t - T qg T qf - T qg ##EQU00010##
and wherein T.sub.pf is the second time and T.sub.pg is the first
time.
[0019] In yet another aspect of the present concepts, a method of
assessing an effect of a modifier on blood coagulation includes the
acts of driving a first portion of a blood sample at a
substantially constant flow rate through a first plurality of
microchannels formed in a microfluidic device substrate and
measuring a pressure, or a variable correlated with pressure, in at
least one of the first plurality of microchannels while the first
portion of the blood sample is moved through the first plurality of
microchannels at the substantially constant flow rate. The method
further includes the acts of determining a first pressure value at
an initiation of flow of the first portion of the blood sample and
determining a first time at which a second pressure value of the
first portion of the blood sample is determined to be about twice
the determined first pressure value of the first portion of the
blood sample. The method further includes the acts of determining a
second time at which a third pressure value of the first portion of
the blood sample is determined to be about (1+e) times the
determined first pressure value of the first portion of the blood
sample and establishing a coagulation model predictive of channel
occlusion for the first portion of the blood sample using the first
time and the second time, for the first portion of the blood
sample, in the relation
.DELTA. P ( t ) .DELTA. P ( 0 ) = 1 + t - T pg T pf - T pg
##EQU00011##
wherein T.sub.pf is the second time and T.sub.pg is the first time.
The method further includes the acts of driving a second portion of
the blood sample at a substantially constant flow rate through a
second plurality of microchannels formed in the microfluidic device
substrate or another microfluidic device substrate and adding a
modifier to one of the second portion of the blood sample or the
second plurality of microchannels. The method further includes the
acts of measuring a pressure, or a variable correlated with
pressure, in at least one of the second plurality of microchannels
while the second portion of the blood sample is moved through the
second plurality of microchannels at the substantially constant
flow rate, determining a first pressure value at an initiation of
flow of the second portion of the blood sample, and determining a
first time at which a second pressure value of the second portion
of the blood sample is determined to be about twice the determined
first pressure value of the second portion of the blood sample. The
method further includes the acts of determining a second time at
which a third pressure value of the second portion of the blood
sample is determined to be about (1+e) times the determined first
pressure value of the second portion of the blood sample and
establishing a coagulation model predictive of channel occlusion
for the second portion of the blood sample using the first time and
the second time, for the second portion of the blood sample, in the
relation
.DELTA. P ( t ) .DELTA. P ( 0 ) = 1 + t - T pg T pf - T pg
##EQU00012##
wherein T.sub.pf is the second time and T.sub.pg is the first time.
The method further includes the acts of comparing the coagulation
model predictive of channel occlusion for the first portion of the
blood sample to the coagulation model predictive of channel
occlusion for the second portion of the blood sample to determine
an effect of the modifier.
[0020] In another aspect of the present concepts, a method of
assessing an effect of a modifier on blood coagulation includes the
acts of driving a first portion of a blood sample at a
substantially constant pressure through a first plurality of
microchannels formed in a microfluidic device substrate and
measuring a flow rate, or a variable correlated with flow rate, in
at least one of the first plurality of microchannels while the
first portion of the blood sample is moved through the first
plurality of microchannels at the substantially constant pressure.
The method further includes the acts of determining a first flow
rate value at an initiation of flow of the first portion of the
blood sample, determining a first time at which a second flow rate
value of the first portion of the blood sample is determined to be
about twice the determined first flow rate value, and determining a
second time at which a third flow rate value of the first portion
of the blood sample is determined to be about (1+e) times the
determined first flow rate value. The method further includes the
act of establishing a first coagulation model predictive of channel
occlusion for the first portion of the blood sample using the first
time and the second time in the relation
Q ( t ) Q ( 0 ) = 1 1 + t - T qg T qf - T qg ##EQU00013##
wherein T.sub.qf is the second time and T.sub.qg is the first time.
The method further includes the act of driving a second portion of
the blood sample at a substantially constant pressure through a
second plurality of microchannels formed in the microfluidic device
substrate or another microfluidic device substrate and adding a
modifier to one of the second portion of the blood sample or the
second plurality of microchannels. The method further includes the
acts of measuring a flow rate, or a variable correlated with flow
rate, in at least one of the second plurality of microchannels
while the second portion of the blood sample is moved through the
second plurality of microchannels at the substantially constant
pressure and determining a first flow rate value at an initiation
of flow of the second portion of the blood sample. The method
further includes the acts of determining a first time at which a
second flow rate value of the second portion of the blood sample is
determined to be about twice the determined first flow rate value
of the second portion of the blood sample and determining a second
time at which a third flow rate value of the second portion of the
blood sample is determined to be about (1+e) times the determined
first flow rate value of the second portion of the blood sample.
The method further includes the act of establishing a second
coagulation model predictive of channel occlusion for the second
portion of the blood sample using the first time and the second
time, for the second portion of the blood sample, in the
relation
Q ( t ) Q ( 0 ) = 1 1 + t - T qg T qf - T qg ##EQU00014## [0021]
wherein T.sub.qf is the second time and T.sub.qg is the first time.
The method further includes the act of comparing the coagulation
model predictive of channel occlusion for the first portion of the
blood sample to the coagulation model predictive of channel
occlusion for the second portion of the blood sample to determine
an effect of the modifier.
[0022] Additional aspects of the invention will be apparent to
those of ordinary skill in the art in view of the detailed
description of various embodiments, which is made with reference to
the drawings, a brief description of which is provided below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows a representation of microfluidic coagulation
device in accord with at least some aspects of the present
concepts.
[0024] FIGS. 2A-2B show dynamics of thrombus formation using
fluorescence timelapse microscopy of labeled fibrinogen, in accord
with at least some aspects of the present concepts, utilizing the
microfluidic coagulation device of FIG. 1.
[0025] FIGS. 3A-3D show a quantitative assessment of whole blood
coagulation in a microfluidic device in accord with FIG. 1 operated
under infusion or pressure pump mode, in accord with at least some
aspects of the present concepts.
[0026] FIGS. 4A-4D show clotting times derived from the
phenomenological analytical models as a function of unfractionated
heparin concentration and shear rate, in accord with at least some
aspects of the present concepts.
[0027] FIGS. 5A-5C show an analysis of platelet aggregation using
the microfluidic coagulation measurement device in accord with at
least some aspects of the present concepts.
[0028] FIG. 6 is a table showing a Goodness of Fit parameter,
R.sup.2, of respective curve fits from derived mathematical
relations for clotting dynamics, Eq. (1) and Eq. (2), at various
shear rates and heparin concentrations for both infusion and
pressure mode of device operation in accord with at least some
aspects of the present concepts.
[0029] FIG. 7 is a table showing a Goodness of Fit parameter,
R.sup.2, of respective curve fits from derived mathematical
relations for clotting times, at various shear rates and heparin
concentrations for both infusion and pressure mode of device
operation in accord with at least some aspects of the present
concepts.
[0030] FIG. 8 is a table showing mean best-fit values of parameters
of the mathematical model for clotting time vs. heparin
concentration in accord with at least some aspects of the present
concepts.
[0031] FIG. 9 is a table showing mean best-fit values of parameters
of the mathematical model for clotting time vs. shear rate in
accord with at least some aspects of the present concepts.
[0032] FIG. 10 shows an analytical model for quantitative
assessment of whole blood coagulation on a microfluidic device
operating in infusion pump mode or pressure pump mode in accord
with at least some aspects of the present concepts wherein, in
infusion mode, the decay in flow follows a sigmoid trend and
wherein, in pressure mode, the pressure grows exponentially, with
the clotting times able to be extracted by fitting the equations of
the analytical model to these measurements respectively.
[0033] FIG. 11 shows whole blood, drawn in sodium citrate, perfused
through the microfluidic device of FIG. 1, and .DELTA.P-Q response
curve is plotted to estimate .DELTA.P(0) and Q(0) in infusion mode
and pressure mode of operation respectively in accord with at least
some aspects of the present concepts.
[0034] FIG. 12 shows an analytical model to predict the clotting
time of whole blood in the coagulation monitoring microfluidic
device as a function of shear rate/stress in accord with at least
some aspects of the present concepts.
[0035] FIGS. 13A-13C shows computational modeling of blood flow in
a biomimetic vascular network, showing shear rate and shear rate
gradients therein, in accord with at least some aspects of the
present concepts.
[0036] FIG. 14 shows the effect of shear and shear gradient on
fibrin and platelet adhesion, wherein it is shown that fibrin and
platelet adhesion is maximum at post-stenotic/diverging section of
the microfluidic device represented in FIGS. 13A-13C.
[0037] FIG. 15 shows that a collagen coating utilized with the
microfluidic device represented in FIGS. 13A-13C reduces clot
monitoring assay time to within 2-20 minutes.
[0038] While this invention is susceptible of embodiment in many
different forms, there is shown in the drawings and will herein be
described in detail preferred embodiments of the invention with the
understanding that the present disclosure is to be considered as an
exemplification of the principles of the invention and is not
intended to limit the broad aspect of the invention to the
embodiments illustrated.
DETAILED DESCRIPTION OF THE INVENTION
[0039] As discussed herein, the inventors found that clotting
dynamics were sigmoidal and, based on this observation, the
inventors developed and experimentally validated phenomenological
mathematical models that accurately predict the temporal dynamics
of whole blood clotting for both injection modes when either
anticoagulant (heparin) concentration or applied shear rate is
varied. The developed models reveal two clotting times: one
indicates when clotting initiates, and the other determines when
full vascular occlusion is produced. These models also incorporate
the biophysical effects of fibrinogen diffusive transport and
platelet aggregation. The inventors demonstrated, further, that the
microfluidic device can, for example, be integrated with automated
fluorescence imaging to enable simultaneous quantitation of
coagulation and platelet aggregation. Accordingly, the
multifunctional coagulation analysis system described herein is
able to provide a quantitative standard for monitoring coagulation
in a patient or subject (e.g., animal or human), either at the
bedside or in a laboratory.
[0040] Described herein, in one embodiment of the present concepts,
is a microfluidic device 100, shown by way of example in FIG. 1,
containing a plurality of parallel microchannels 110 that mimic 125
.mu.m diameter blood vessels and permit real-time analysis of
clotting dynamics when a small volume (2-5 mL) of whole human blood
is infused under either constant flow or constant pressure. The
microfluidic device 100 used to obtain the data described herein
comprises 12 parallel lanes of 200 .mu.m wide and 75 .mu.m high
channels that repeatedly turn 60.degree. over a length of 50 mm, as
shown in FIG. 1, to provide a high hydrodynamic resistance, but
retain laminar flow.
[0041] As discussed below, in the test configuration, 2-5 ml of
human whole blood was pumped through an infusion or pressure pump
followed by an inline pressure sensor that connected to ports 120a,
120b of the microfluidic device 100 via appropriate medical grade
tubing (e.g., 1.6 mm ID). In infusion mode, clot formation leads to
an increase in pressure, whereas in constant pressure mode, the
flow rate drops with respect to time. In analyzing these results,
phenomenological mathematical models and fit regression curves were
applied to changes in pressure or flow, respectively. The
characteristic constants of these models indicate clotting times at
two stages--when the clot formation is accelerating and when the
clot has completely occluded the channel. The clotting times are
determined by the concentration of the anti-coagulant (e.g.,
heparin) and applied shear. Fluorescense microscopy of fibrinogen
and platelets allow real-time monitoring of fibrin formation and
platelet aggregation simultaneously.
[0042] In the testing conducted with the microfluidic device 100 of
FIG. 1, fresh human blood stored in a 5 ml syringe (slip-tip
Plastipak, BD, Franklin Lakes, N.J.) was pushed via syringe pump
(PHD Ultra CP, Harvard Apparatus, Holliston, Mass.) through an
inline, disposable pressure sensor (PREPS-N-000, PendoTECH,
Princeton, N.J.) followed by the PDMS device within 15 minutes of
blood draw. When flow rate was maintained to be constant, the
syringe pump was operated in `infusion` mode and the channel
occlusion was measured by recording the rise in pressure over time
using a data acquisition and analysis software (Winwedge Pro,
TALtech, Philadelphia, Pa.). In the constant pressure operation
mode, the syringe pump was used in `pressure` mode that processes
the feedback from a pressure sensor and modulates the motor speed
and flow rate to maintain a set pressure. 2.5 inches and 6 inches
of 3.2 mm OD, 1.58 mm ID medical grade tubing (Tygon S-50-HL, Saint
Gobain Plastics, Merrimack, N.H.) were connected to the ports 120a,
120b of the device. As configured, one end of the inlet side tubing
125 is connected to the pressure sensor and the outlet side of the
tubing is dipped in 3.8% sodium citrate. To further reduce clot
formation inside the syringe, sensor and tubing, the
blood-contacting surfaces were treated, if required, with slippery
liquid-infused porous surface (SLIPS) technology, wherein
low-pressure radio-frequency plasma exposure was used to activate
the surfaces, followed by covalent coupling of an inert silane
layer and addition of medical-grade liquid perfluorocarbon, used in
blood substitutes. The syringe in the syringe pump was manually
agitated every 2-3 minutes to prevent sedimentation of erythrocytes
in the blood. Thrombus formation was observed using time-lapse
imaging of an imaging device 130 of fluorescently-labeled
fibrinogen (150 mg/ml, Alexa Fluor 488, Invitrogen, Grand Island,
N.Y.). In the test setup, the imaging device 130 used was a
fluorescence imaging configured Carl Zeiss Axio 3 Observer
microscope (10.times., NA 0.3 objective), but other imaging devices
may be readily used in accord with the present concepts.
[0043] Platelets were isolated from whole blood by two
centrifugations (200 g followed by 500 g) and labeled with calcein
orange (2 .mu.M, Invitrogen, Grand island, N.Y.) for 10 minutes.
Multiple fluorescent microscopic images were recorded from
neighboring regions using automatic scanning and stitched together
to form a large region panorama. The presence and size of large
fluorescent platelet aggregates were then analyzed using automated
image capture and an image analysis protocol. The heparin
concentration and wall shear rate were varied independently in
these experiments. The wall shear stress/rate was determined from
analytical formulae derived for the rectangular microchannels
110.
[0044] The microfluidic device 100 (FIG. 1) used in the testing
described herein comprises SU8 2075 (MicroChem. Corp., Newton,
Mass.) master templates fabricated on Si wafers (University Wafer
Corp., Boston, Mass.) using photolithography, and more particularly
soft lithography of polydimethylsiloxane (PDMS). 27 Slygard 184
PDMS prepolymer (Dow Corning, Midland, Mich.) was cast on the
silanized master, which had the positive relief of the channel
features formed by the SU-8 photoresist. The PDMS was then cured at
60.degree. C. in a convection oven for 120 minutes. The cured PDMS
was peeled off the master and bonded to a 500 .mu.m high PDMS
coated glass slide after treating both with oxygen plasma (Plasma
Etch, Carson City, Nev.). The microfluidic devices 100 was primed
with perfluorodecalin (PFD, Sigma-Aldrich), a medically approved
lubricant, before use.
[0045] After informed, written consent as per ethical guidelines of
Institutional Review Board (IRB) of Partners Healthcare and Harvard
University, blood samples were collected from non-smoking healthy
volunteers in a standard 6 ml no-additive blood vacutainer (BD and
Company, Franklin Lakes, N.J., USA) and 1000 U/ml unfractionated
heparin was immediately added to a required concentration.
Coagulation experiments were initiated within .about.15 minutes
after the blood draw. For experiments where heparin concentration
was below 0.25 U/ml, blood was first drawn in 3.2% 5 ml sodium
citrate vacutainers (BD and Company, Franklin Lakes, N.J., USA).
Citrated blood (Research Blood Components, Brighton, Mass., USA)
was also purchased for some studies, and the coagulation activity
of these samples was restored by adding 75 .mu.l/ml of 100 mM
calcium chloride/75 mM magnesium chloride solution. Aspirin (Sigma
Aldrich, St. Louis, Mo.) was dissolved in phosphate buffer solution
(PBS) to a concentration of 20 mM and added to blood to reach a
final concentration of 500 .mu.M. Prasugrel (Sigma Aldrich, St.
Louis, Mo.) was dissolved in dimethylsulfoxide (DMSO) at 1 mg/ml
and added to blood to reach a final concentration of 25
.mu.g/ml.
[0046] In the statistical analyses described herein, unless
otherwise specifically mentioned, all data is presented as
mean.+-.SD. Two-tailed P values were obtained from the statistical
t-test to compare the means. Data analysis and curve fitting was
performed using Graphpad Prism V6 (Graphpad Software, San Diego,
Calif.).
[0047] FIGS. 2A-2B show dynamics of thrombus formation using
fluorescence timelapse microscopy of labeled fibrinogen, in accord
with one aspect of the present concepts, utilizing the microfluidic
coagulation device 100 of FIG. 1. In FIG. 2A, the fluorescence
intensity normalized by the maximum, follows a sigmoidal trend over
time in a microchannel 110, leading to its occlusion. The data can
be fit using a three-parameter sigmoid equation. The dotted lines
and shaded grey area show 95% confidence interval of the fitted
curve. n=3, R.sup.2=0.99. In FIG. 2B, stacked fluorescent
micrographs of labeled fibrinogen at shown at four different time
durations from timelapse imaging of fluorescent fibrinogen for
whole blood flow containing 0.75 U/ml heparin anti-coagulant and an
imposed shear rate of 1250 sec-1. Each figure is a scan of
4.times.1 tiles (10.times.objective) stitched together. The white
scale bar at the bottom right of the bottom micrographs is 500
.mu.m.
[0048] FIGS. 3A-3D show a quantitative assessment of whole blood
coagulation in a microfluidic device 100 in accord with FIG. 1
operated under infusion or pressure pump mode. By varying the
heparin concentration at a given shear rate/stress of 350
sec.sup.-1 (14 dynes/cm.sup.2), in infusion mode, the pressure
grows exponentially with time (FIG. 3A) and in pressure mode, the
flow drops in sigmoidal fashion with time (FIG. 3B). In FIGS.
3A-3B, reference numeral 300a denotes a plot corresponding to a
flow rate of 0 U/ml, reference numeral 300b denotes a plot
corresponding to a flow rate of 0.25 U/ml, reference numeral 300c
denotes a plot corresponding to a flow rate of 0.5 U/ml, and
reference numeral 300d denotes a plot corresponding to a flow rate
of 1 U/ml. Similarly, by varying the shear rate/stress at heparin
concentration of 0.5 U/ml, in infusion mode, the pressure grows
exponentially with time (FIG. 3C) and, in pressure mode, the flow
drops in sigmoidal fashion with time (FIG. 3D). In FIGS. 3C-3D,
reference numeral 310a denotes a plot corresponding to a flow rate
of 150 sec.sup.-1, reference numeral 310b denotes a plot
corresponding to a flow rate of 350 sec.sup.-1, reference numeral
310c denotes a plot corresponding to a flow rate of 750 sec.sup.-1,
and reference numeral 310d denotes a plot corresponding to a flow
rate of 1500 sec.sup.-1. The solid lines in FIGS. 3A-3D are
measured quantities and the dotted lines represent a 95% confidence
interval of regression curves fit with analytical model Eq. (1) and
Eq. (2), described herein, respectively. The goodness of fit
parameter R.sup.2 is tabulated in FIG. 6.
[0049] FIGS. 4A-4D show clotting times derived from the
phenomenological analytical models as a function of unfractionated
heparin concentration and shear rate, in accord with at least some
aspects of the present concepts. By varying the heparin
concentration in the range 0-1 U/ml and setting the shear rates 350
sec.sup.-1 (14 dynes/cm.sup.2) and 1250 sec.sup.-1 (50
dynes/cm.sup.2) respectively, in infusion mode (FIG. 4A), the
clotting times, (T.sub.pg and T.sub.pf), and in pressure mode (FIG.
4B), the clotting times, (T.sub.qg and T.sub.qf), increase
exponentially with heparin concentration. In FIGS. 4A-4B, reference
numeral 400a denotes a plot corresponding to T.sub.pg, T.sub.qg at
350 sec.sup.-1, reference numeral 400b denotes a plot corresponding
to T.sub.pf, T.sub.qf at 350 sec.sup.-1, reference numeral 400c
denotes a plot corresponding to T.sub.pg, T.sub.qg at 1250
sec.sup.-1, and reference numeral 400d denotes a plot corresponding
to T.sub.pf, T.sub.qf at 1250 sec.sup.-1. By varying the shear
rates in the range 75-2500 sec.sup.-1 (3-100 dynes/cm.sup.2) and
setting the heparin concentration 0.25 and 0.5 U/ml respectively,
in infusion mode (FIG. 4C), the clotting time times, (T.sub.pg and
T.sub.pf), decay exponentially and in pressure mode (FIG. 4D), the
clotting time times, (T.sub.qg and T.sub.qf), follow the relation,
Clot Time=Z0.gamma..sup..omega.e.sup.-.phi..gamma., with shear
rate/stress. In FIGS. 4C-4D, reference numeral 410a denotes a plot
corresponding to T.sub.pg, T.sub.qg at 0.25 U/ml, reference numeral
410b denotes a plot corresponding to T.sub.pf, T.sub.qf at 0.25
U/ml, reference numeral 410c denotes a plot corresponding to
T.sub.pg, T.sub.qg at 0.5 U/ml, and reference numeral 410d denotes
a plot corresponding to T.sub.pf, T.sub.qf at 0.5 U/ml. The dotted
lines are regression curves fitted to the analytical relationships
respectively wherein n=3 experiments were conducted in each case
and the R.sup.2 goodness of fit parameter for each case is
tabulated in FIG. 7.
[0050] FIGS. 5A-5C show an analysis of platelet aggregation using
the microfluidic coagulation measurement device 100 in accord with
at least some aspects of the present concepts. In FIG. 5A,
fluorescent stacked images of the microfluidic device 100 show
formation of aggregates of fluorescently-labeled platelets when
whole human blood is flowed through the microfluidic device of FIG.
1 without (Control) or with 500 .mu.M aspirin and 25 .mu.g/ml
prasugrel (Drugs). Images at the center of FIG. 5A are higher
magnification insets of the upper and lower images. The scale bar
(white at top right of top slide in FIG. 5) is 5 mm. FIG. 5B shows
addition of the platelet inhibitor drug combination (Drugs)
increased the clotting time compared to Control when the device was
operated at a constant shear rate of 1250 sec.sup.-1 (n=4), wherein
the solid circle represents the Control and the empty circle (right
side of the line segments) represents the Drugs. In FIG. 5C,
addition of the drug combination significantly decreased the number
of platelet aggregates in the blood compared to control untreated
samples (*P<0.05; n=5).
[0051] The microfluidic coagulation device 100 was designed to
operate at shear rates ranging from about 75 sec.sup.-1 to 2500
sec.sup.-1 (3 to 100 dynes/cm.sup.2) in such a way that the
corresponding flow rates can be maintained within the range of
about 5-150 Desirably, a shear rate is maintained to be
substantially constant, so as not to vary more than a predetermined
amount from the set shear rate (e.g., within 10% from a set value,
and still more preferably within about 5% from the set value, and
still more preferably within 2% from the set value). Flow rates
outside of the above-noted range, for the particular microfluidic
coagulation device 100 utilized, were found to be undesirable as
they would result in frequent red blood cell sedimentation or
require large volume of human donor blood respectively. However,
different microchannels 110 configurations (e.g., different
cross-sectional area) and/or different clinical parameters (e.g., a
larger volume of patient blood) could certainly warrant flow rates
outside of the above-specified range in order to operate at shear
rates ranging from about 75 sec.sup.-1 to 5000 sec.sup.-1 (3 to 200
dynes/cm.sup.2). By way of example, microchannel 110 configurations
in the microfluidic devices 100 of FIG. 13A and FIG. 14 provide a
geometry that allows the fluid to pass through a converging zone
500 (flow acceleration; prestenosis) into multiple lanes made of
microchannels of substantially constant cross-sectional area 520
having curved sections and straight sections. The fluid then exits
through a diverging section 540 (flow deceleration; post-stenosis)
into a common outlet. The straight section microchannels 110 of the
microfluidic device 100 were sized to enable real-time optical
microscopic imaging using a low magnification (10.times.; 0.3 N.A.)
objective. For these practical reasons, the microfluidic device 100
of FIG. 1 comprises 12 parallel microchannels 110 that are each
approximately 200 .mu.m wide and 75 .mu.m high. The rectangular
cross-sectional surface area of the microchannels 110 was
equivalent to a 125 .mu.m diameter circular arteriole. To acquire a
good signal-to-noise ratio from the pressure sensor utilized in the
set-up of FIG. 1, the channel length was optimized for the highest
possible hydrodynamic resistance, while restricting the overall
length of the microfluidic device 100 so that it fits on a standard
glass microscope slide (50 mm.times.75 mm). This was achieved by
incorporating a continuous series of 60.degree. bends in the
channels, except a straight 1 cm section at the center of the
device where optical imaging is performed (FIG. 1). The particular
curvilinear structure of the microchannels 110 was designed, given
the arbitrary geometric constraint of utilizing a standard glass
slide, to achieve the best signal-to-noise ratio out of the sensor
used (e.g., the pressure sensor used to measure pressure), that is,
it can measure smaller changes in hydraulic resistance. If a more
expensive, or more highly-sensitive, pressure sensor were used, for
example, the design-envelope for the microchannels 110 changes,
permitting different geometries (e.g., straight) to be used with a
suitable signal-to-noise ratio.
[0052] Absent the mere preference to size the microfluidic device
100 to fit on a standard glass microscope slide, desired
constraints of low Reynolds number flow and a high signal-to-noise
ratio (e.g., imposing a minimum dimension of the microchannels of
about 15 .mu.m and a maximum dimension of about 1.5-2.0 mm), there
is no limit on the sizing of the microfluidic device 100 and/or
microchannels 110. Thus, the microfluidic device 100 may be
dimensionally larger or smaller than that described herein and,
further, the number or microchannels 110 and/or microchannel
configurations may be freely varied (e.g., the microchannels 110
may be straight, may be defined by a 3-D geometry, etc.). Further,
although not shown, one or more of the microchannels 110 may
optionally comprise one or more additional microchannel inlets
permitting the any of the one or more microchannels to have
additional fluid(s) introduced therein, such as a drug to be mixed
in with the flow in the microchannel). Moreover, it is to be
emphasized that the depicted microfluidic device 100 was designed
in support of the testing and analysis described herein and the
utilization of microscopy or imaging devices (e.g., 130, FIG. 1) is
not a necessary aspect of the microfluidic device-based coagulation
system described herein. Due to the relationships derived by the
present inventors, described herein, the dynamics of coagulation
are determinable solely by measurement of pressure or flow rate.
Accordingly, whereas the disclosed microfluidic device 100 of FIG.
1 comprises a transparent substrate to facilitate imaging of the
coagulation, an opaque substrate alternatively may be used. Only if
platelet aggregration is simultaneously measured, microscopy is
needed.
[0053] The dynamics of fibrin clot formation in a blood vessel in
vivo, or in an in vitro hollow channel, consist of three stages--a
steady reaction time, a growth phase, and saturation (full
stenosis)--resulting in a sigmoid curve. To validate the sigmoidal
dynamics of clot formation in the microfluidic device 100 of FIG.
1, comprising rectangular channels, time-lapse microscopic analysis
of whole human blood spiked with fluorescently-labeled fibrinogen
and an intermediate level therapeutic dose (0.75 U/mL) of heparin
anticoagulant was performed while imposing a shear rate of 1250
sec.sup.-1 (50 dynes/cm.sup.2) in constant flow mode. When the mean
fluorescence intensity, I(t), normalized by the intensity of a
fully clotted region (I.sub.max) was plotted against time, the
inventors determined that, at a fixed site within the linear
portion of the microfluidic channel, clotting followed a sigmoidal
trend indicative of the three stages of clot formation (FIGS.
2A,B).
[0054] Previous studies have shown that the size of a growing
thrombus measured in vitro correlates linearly with measured light
intensity. Therefore, it was assumed that, in the microfluidic
device 100, I(t)/I.sub.max.apprxeq.A.sub.max/A(t) , where A(t) is
the cross-sectional area available for blood flow through the
occluding channel at a given time, and A.sub.max is the initial
cross-sectional area of the microchannel 110.
[0055] The hydraulic resistance (R.sub.h) of the occluding
microchannel 110 approximately scales as
R h ( t ) .apprxeq. 1 A ( t ) 2 . ##EQU00015##
Because the microfluidic device 100 of FIG. 1 has parallel
microchannels 110 and each microchannel will not occlude equally at
the same time, the hydraulic resistance of the whole device scales
as
R h ( t ) .apprxeq. 1 A ( t ) k , k .ltoreq. 2. ##EQU00016##
[0056] For simplicity, it is assumed that k is unity, which leads
to the conclusion that R.sub.h(t) of an occluding microchannel 110
follows a simple sigmoid. Based on the Hagen-Poiseuille law
(Q=.DELTA.P/R.sub.h) of laminar flow, where Q is flow rate and
.DELTA.P is pressure-drop, when constant flow is applied with a
standard syringe pump using the infusion mode of operation
(Q=constant), the present inventors proposed the following model to
predict clotting times and clotting dynamics (e.g., channel
occlusion) where .DELTA.P increase follows the following relation
(FIG. S1):
.DELTA. P ( t ) .DELTA. P ( 0 ) = 1 + t - T pg T pf - T pg ; Q ( t
) = Q ( 0 ) ( 1 ) ##EQU00017##
where T.sub.pg and T.sub.pf are the characteristic parameters of
the fitted exponential growth curve that represent time for the
pressure to double and (1+e) times its initial value, respectively.
Physiologically, these times represent the growth and saturation
phases of clotting under constant flow, respectively, which is
analogous to development of hypertensive pressures in an arterial
vessel in vivo.
[0057] Alternatively, extending the analytical model for predicting
clotting times and clotting dynamics to blood injection with a
pressure pump (.DELTA.P=constant), the present inventors predicted
that the drop in flow (Q) of an occluding channel follow the
following relation of an inverted sigmoid (FIG. S1):
Q ( t ) Q ( 0 ) = 1 1 + t - T qg T qf - T qg ; .DELTA. P ( t ) =
.DELTA. P ( 0 ) ( 2 ) ##EQU00018##
wherein T.sub.qg and T.sub.qf are the characteristic parameters of
the fitted sigmoidal decay curve that represent time for flow to
reduce to half and (1+e) times its initial value, respectively.
Physiologically, these times respectively represent the growth and
saturation phases of clotting, which could lead to flow stasis as
might occur, for example, in a blood vessel of the venous
circulation in vivo.
[0058] To determine the initial values of the mathematical models,
.DELTA.P(0) and Q(0), the present inventors assumed that the
physical parameters of the blood were constant at the beginning of
coagulation monitoring, and therefore, can be obtained from the
.DELTA.P-Q calibration curve of citrated blood perfused through the
device at different flow rates. The data obtained indeed revealed a
linear relationship between the change in pressure and applied flow
rate for laminar anti-coagulated blood flow and therefore, the line
of linear regression represents the .DELTA.P-Q calibration curve
(FIG. 11). The rise in pressure when the flow rate is constant
(FIG. 3A) and decay in flow when the pressure is constant (FIG. 3B)
were experimentally determined at different heparin concentrations
at a constant physiological shear rate of 350 sec.sup.-1(14
dynes/cm.sup.2) and it was found that the regression curves based
on mathematical models (1) and (2) fit the data with a high level
of accuracy (FIG. 6). Similarly, when the fluid shear stress at a
constant heparin concentration (0.5 U/mL) was varied, it was found
to fit the above analytical models to the experimental measurements
for pressure rise at a constant flow rate (FIG. 3C) or flow decay
at a constant pressure (FIG. 3D), with the curve fits being found
to be extremely accurate (FIG. 6). The goodness of fit parameter
(R.sup.2) values shown in FIG. 6 from the entire regression curve
fits also confirmed that the mathematical models are highly
reliable. Therefore, the best-fit values of model parameters, that
is, clotting times (T.sub.pg and T.sub.pf, T.sub.qg and T.sub.qf)
represent the clotting dynamics of human whole blood in this
biomimetic microfluidic device 100 for heparin concentrations in
the range 0-1 U/ml and shear rate in the range 75-2500 sec.sup.-1
(6-100 dyne/cm.sup.2).
[0059] The inventors then set out to test the diagnostic and
clinical utility of blood clotting times (T.sub.pg and T.sub.pf for
infusion; T.sub.qg and T.sub.qf for pressure) determined from these
regression models by validating their response to changes in
unfractionated heparin concentration and applied shear, and
comparing these results with those from known systems. The standard
coagulation tests, including activated partial thromboplastin time
(APTT) and activated clotting time (ACT), have shown an exponential
relation between clotting time and the concentration of
unfractionated heparin when tested at clinically relevant
concentrations (0-1 U/mL). This relation can be described as,
ClotTime=T0e.sup..tau.C.sup.h, where T0 is the clotting time for
blood with no heparin and .tau. is the heparin sensitivity value.
To validate this exponential relationship and evaluate the utility
of microfluidic device 100 for analyzing anti-coagulation therapy
in the clinic, heparin sensitivity in the relevant concentration
range (0-1 U/mL) was measured and the characteristic clotting
parameters, T.sub.pg and T.sub.pf (infusion mode) and T.sub.qg and
T.sub.qf (pressure mode) were evaluated, as determined by fitting
regression curves based on equations (1) and (2), respectively, for
coagulating whole human blood flow in the microfluidic device 100,
as described above. In the infusion mode, both T.sub.pg and
T.sub.pf accurately exhibited exponential increases as the heparin
concentration was raised from 0 to 1 U/mL at physiological (350
sec.sup.-1; 14 dynes/cm.sup.2) and pathological (1250 sec.sup.-1;
50 dynes/cm.sup.2) fluid shear levels as reported by the goodness
of fitness parameter (FIG. 4A, FIG. 7). The exponential trend using
the constant pressure mode was also validated. Both T.sub.qg and
T.sub.qf increased exponentially as the heparin concentration was
raised using the same two shear rates (FIG. 4B, FIG. 7), and in all
cases, clotting times were reduced at high versus low shear (FIGS.
4. A,B). In addition, the model predicts a clotting time of blood
from a healthy patient with no-anticoagulant (T0) to be
approximately 2-12 minutes, and the heparin sensitivity value
(.tau.) to be in the range of 1.75-3.5 (U/ml).sup.-1, which is
consistent with typical values reported by the standard coagulation
tests, shown in FIG. 8.
[0060] Current coagulation tests applied in the clinic do not
incorporate the physiological contributions of hemodynamic shear
stresses that result, for example, in increased clotting of veins
at low shear stresses (relative to physiological) and in small
arteries at high shear stresses. In addition, standard clotting
time instruments report only one value of clotting time at stasis
that could be inaccurate in patients undergoing procedures
employing extra-corporeal circuits (e.g., ECMO, dialysis,
hemofiltration) where blood flow is both variable and a critical
determinant of coagulation. Thus, the present inventors set out,
using microfluidic device 100, to develop and test a mathematical
model to predict the clotting time when shear is varied. Clotting
at low shear is governed by the diffusion-reaction transport of
coagulation factors such as fibrinogen and therefore, a model of
power-law kinetics (.gamma..sup..omega.) was used, where .omega. is
the power-law constant. Coagulation at high shear is dominated by
platelet activation, the impact of which on blood clotting time was
modeled using an exponential relationship (e.sup.-.phi..gamma.),
where .phi. is the decay constant. For simplicity, mutual
independence of fibrinogen diffusion and platelet aggregation was
assumed, and the inventors developed a mathematical model of
clotting time in which Clot
Time=Z0.gamma..sup..omega.e.sup.-.phi..gamma., where Z0 is a model
constant (the individual and coupled behavior of the power-law and
exponential terms of this analytical model are shown in FIG.
8).
[0061] When blood clotting was measured using in the infusion mode,
a decrease in clotting times (T.sub.pg and T.sub.pf) was only
observed when shear rates were increased from about 150 sec.sup.-1
to about 2500 sec.sup.-1 (about 6 to 100 dynes cm.sup.2) (FIG. 4C).
In the infusion mode, when shear rate was reduced to 75 sec.sup.-1,
the blood did not clot, as no increase in pressure was observed
during the 60 minute infusion time. Accordingly, for this
combination of microfluidic device 100 (FIG. 1) and test
conditions, the inventors were not able to fit the mathematical
model accurately or extract clotting times. Because a drop in
clotting time at the lowest shear rates that could be applied in
the test configuration were not observed, the contribution of
diffusive transport of coagulation enzymes proposed in the
mathematical model appears to be negligible in this device at shear
rates above 75 sec.sup.-1. Thus, by assuming the power constant
(.omega..apprxeq.0), clotting times can be fit accurately with a
more simple exponential decay relation:
ClotTime=Z0e.sup.-.phi..gamma. (FIGS. 7, 9).
[0062] However, when similar measurements were made in the constant
pressure mode, the clotting times (T.sub.qg and T.sub.qf) decreased
at shear rates below 350 sec.sup.-1 (14 dynes/cm.sup.2) for two
different heparin concentrations (0.25 U/ml and 0.5 U/ml; FIG. 4D).
At shear above 350 sec.sup.-1, an exponential decrease was observed
and our mathematical model fit accurately with the power constant,
.omega.>0 (FIGS. 7, 9). Interestingly, the model also predicted
that the maximum clotting time will occur at a shear rate between
200 to 500 sec.sup.-1 (8-20 dynes/cm.sup.2), which corresponds well
to the physiological shear range expected for a small (.about.100
.mu.m) blood vessel. These results suggest that when studied in the
constant pressure mode of operation, blood clotting inside the
microfluidic device 100 can be determined by diffusion of
coagulation proteins at low shear. In addition, the model predicts
that platelet aggregation can significantly contribute to
thrombosis at high shear in both operating modes (FIG. 9).
[0063] Platelets have a significant role in thrombus formation and
one of the advantages of this microfluidic device 100 is that it
also can be optionally integrated with automated optical microscopy
to quantify platelet aggregation for lab diagnostics and to guide
anti-platelet therapy in the clinic. As proof of concept, two
platelet activation inhibitors, aspirin (500 .mu.M) and prasugrel
(25 .mu.g/ml), were added to recalcified citrated human blood along
with fluorescently-labeled autologous platelets; these two drugs
are used clinically to treat acute coronary syndromes. The blood
was then pumped through the microchannels 110 of the microfluidic
device 100 at a constant shear rate of 1250 sec.sup.-1 (50
dynes/cm.sup.2), and scanned a region (24.8.times.7.5 mm) of one
microchannel 110 immediately after occlusion was detected (FIGS.
5A,B). In each experiment performed (n=4), the treatment with this
drug combination increased the clotting times, T.sub.pg and
T.sub.pf, which is consistent with past studies using other
clotting assays. On average, the clotting times increased by 13.02%
and 6.79%, respectively, in the microfluidic device 100 (FIG. 5C).
These studies also revealed that the number of large platelet
aggregates was reduced by about 45% (p<0.05, n=5) due to the
addition of the drug combination (FIG. 5D).
[0064] As is known, the pathophysiology of blood coagulation
involves interplay among blood components, the adhesion surface,
and fluid dynamics, now popularly known as the Virchow's triad.
Clearly, hemostasis is dynamic in nature in that blood-surface
interactions leading to thrombosis and fibrinolysis occur in the
presence of blood flow. More specifically, independent of soluble
clotting agonists, thrombus formation and platelet aggregation is
enhanced due to shear gradients arising from acceleration and
deceleration of flow at stenotic regions and clotting is most
pronounced post-stenosis, where the flow decelerates. However, the
agonists, such as collagen or von Willebrand factor (vWf), may also
contribute in stabilizing the clot.
[0065] Using this microfluidic device 100, distinct shear rates,
gradients of shear and relevant hemodynamics can be created that
permit measurement of normal and abnormal coagulation responses
under more physiological conditions, and this potentially could be
carried out in the hematology laboratory or elsewhere, such as at a
clinic, doctor's office, or patient's bedside. This device is
flexible in operation, for it will afford the physician or
clinician to pre-select key parameters such as blood additives
(anti-coagulants, drugs, activators, etc.), governing shear rate,
microchannel topology (for e.g., a combination of width, height and
length to get desirable surface-to-volume ratio at any fixed shear
rate) and number of independent microchannels to use. These
decisions may be based on the pathology, disease, or condition (if
any are known) under investigation (e.g., deficiency in platelet
function would advantageously indicate operation at a higher shear
since that is where platelets respond most, a suspected venous
thrombosis would advantageously operate at a lower shear to
minimize impact of platelets on the result). In accord with the
present concepts, real-time evolution of blood clots can be
recorded and quantified, which is not possible with the current
gold standard tests. The blood can be pumped in one of two
operating modes (constant pressure or constant flow) that can be
selected to mimic the function of the vasculature in vivo--constant
flow rate or infusion where exponential growth of pressure is
recorded, or alternatively, constant pressure where sigmoidal decay
in flow is recorded. In yet other aspects, it may be possible to
operate the constant pressure and constant modes in conjunction
with one another.
[0066] By applying this biomimetic approach to measuring
coagulation, the present inventors presented and empirically
validated general phenomenological mathematical models that predict
dynamics of thrombus formation in both operating modes of blood
injection (FIGS. 1, 2). Although coagulation biology is complex and
yet to be described completely, it involves non-linear interplay
between multiple pro-coagulation and fibrinolytic factors along
with platelets. Due to this complexity coupled with the influence
of hemodynamic shear forces, an aggregating thrombus can
occasionally break and embolize in vivo, and this can be detected
in the microfluidic device 100 as measured by occasional
fluctuations of growing pressure or decaying flow when blood
coagulates (FIG. 2). However, the analytical models presented
herein, being global, predict the empirical observation accurately
if the data is recorded for the entire clotting process (FIG. 6)
and enable the user to extract relevant characteristic parameters
that quantitatively define the clotting time of blood under these
different physiological conditions (FIG. 3). Unlike current
diagnostic tests that provide only one output as the final clotting
time, the present concepts provide two characteristic clotting
times of the coagulation cascade: time when clotting is actively
accelerating and time when the clot has fully occluded the flow
(FIG. 3).
[0067] The concentration of anticoagulant and the shear rate (and
gradient) are the two major determinants of these clotting times
when measuring and monitoring of thrombogenicity. The inventors
found that the clotting times increase exponentially with the
concentration of unfractionated heparin and its sensitivity values
are consistent with those determined by other standard tests (FIGS.
4A-B, FIG. 8). Thus, the data strongly suggest that the
microfluidic device 100 can be utilized in the clinic as an
alternative and more quantitative instrument for anti-coagulation
monitoring.
[0068] When clotting times were analyzed in response to varying
shear, the accuracy of the disclosed power-exponential mathematical
model (FIG. 7) was confirmed. However, different clotting behaviors
were observed when constant flow and constant pressure mode of
blood injection were utilized (FIGS. 4C-D), which are believed to
be attributed to the fact that under constant flow, local shear and
Reynolds number can rise to much higher levels at sites where clots
form and begin to occlude the channel lumen, as occurs in partially
occluded vessels in vivo. These abnormally high shear stresses also
can induce thrombi and platelet aggregates to break apart and be
released as emboli. The finding that clotting times are higher in
the infusion mode compared to the pressure mode in the shear regime
that is below physiological can therefore be explained if platelet
activation and aggregation do not dominate over embolization at low
shear. Also, at low shear in the constant pressure mode, it is
possible that fibrinogen diffusion is significant, like in venous
blood vessels as the model predicts (FIG. 9). Importantly, this is
in contrast to in vitro clotting assays that lack the ability to
differentiate between venous and arterial thrombosis. Based on
these observations, it is believed that the pressure-driven flow of
whole blood, that mimics in vivo thrombosis better at low shear
rates, can be particularly suitable for monitoring of deep vein
thrombosis (DVT) whereas it is believed the standard infusion-based
syringe pump could can be particularly suitable for monitoring
arterial thrombosis (AT), which may be validated in further testing
(e.g., by analyzing specific biomarkers of these conditions, such
as using D-dimer and neutrophil extracellular traps (NETs), which
are hallmarks of DVT).
[0069] Based on the results disclosed herein, the relationships of
clotting times with respect to heparin concentration and shear can
be combined to produce the following analytical relationship of
clotting time for the infusion mode of pumping blood:
( T pg , T pf ) = A ( T pg , T pf ) B ( T pg , T pf ) C uh - C ( T
pg , T pf ) .gamma. ( 3 ) ##EQU00019##
[0070] Similarly for the pressure mode, the relationship is as
follows:
( T qg , T qf ) = A ( T qg , T qf ) .gamma. .omega. B ( T qg , T qf
) C uh - C ( T qg , T qf ) .gamma. ( 4 ) ##EQU00020##
[0071] The model equations (3) and (4) can fit in equations (1) and
(2), respectively, to determine the anticoagulant- and
shear-dependent temporal dynamics of coagulation independent of
system properties. The constants appearing in these equations (A,
B, C and .omega.) are patient specific and may depend upon blood
properties that can be empirically determined by curve fitting the
analytical equations (1) and/or (2). Thus, the patient-specific
constants in equations (3) and (4) are derived from the pressure
curve and/or flow curve obtained when the patient's blood sample is
allowed to clot and the resulting growth in pressure and/or decay
in flow, as applicable, is fit to equations (1) and/or (2) to
extract clotting times. These clotting times are functions of, for
example, shear and heparin concentration and these parameters,
being known in the test performed, can then permit comparison of
the clotting time extracted to a standard calibrated clotting time
curve (e.g., a baseline curve for the patient, a standard
population curve, etc.). In the clinic, these constants might be
regularly monitored by the clinician to determine the clotting
status of a patient undergoing anti-coagulation therapy or even
routine medical examination. For example, responsive to changes in
a patient, these patient-specific constants can increase or
decrease over time, as these patient-specific constants can be
influenced by or depend on other patient-specific markers (e.g.,
exercise, cholesterol, dietary habits, etc.). Thus, in accord with
the present concepts, the clinician may utilize changes in such
patient-specific constants as a diagnostic tool to relate such
changes to, for example, a clinical manifestation of disease (e.g.,
arteriosclerosis, etc.), a suggestion of a particular
susceptibility, and/or a desirability for additional testing to
better characterize the results. As one example, if a 35-yr old
female patient's "A" constant value is much lower or higher (e.g.,
a 50% decrease or increase) as compared to what would be normally
observed for that patient (or for a selected population sample
inclusive of 35-yr old females), such deviation may correlate to
the a certain malady or susceptibility and follow up tests could be
suggested and/or a therapy determined responsive to such deviation.
In healthy patients, both clotting parameters representing growth
and saturation of clot formation follow similar trends when heparin
concentration or shear rate is varied. However, it is possible that
novel anti-coagulants and anti-platelet drugs produce different
behaviors in clotting dynamics, and measurement of the two clotting
parameters will enhance sensitivity and specificity of diagnosis
and treatment.
[0072] By adding an automated imaging protocol, the microfluidic
device 100 can be extended to simultaneously measure large platelet
aggregates, so it can be used to monitor adjuvant anti-platelet
therapy. This method could also be combined with other microscopy
techniques, such as confocal imaging or on-chip flow cytometry, to
enable more sensitive analysis of platelet activity, and to explore
effects of other platelet activators (e.g., collagen, tissue
factors), thus enabling the analysis of platelet activation led
thrombosis independent of fibrinogen-thrombin led thrombosis. These
phenomenological analytical models can be further advanced by
incorporating influences of other cellular components of blood,
such as leucocytes and erythrocytes, on the clotting times as well.
Being a global and quantitative coagulation test, the microfluidic
device 100, and the relations disclosed here (Eq. (1)-Eq. (4)),
offer a potential way to tackle more complex diseases, such as
sepsis and sickle cell anemia, where other cells (e.g., bacteria
and sickled erythrocytes) also contribute to the coagulation
response. Further, the thrombus monitoring device can be operated,
ex vivo, by directly attaching it to catheters or extra-corporeal
devices and thus enabling analysis if native blood not drawn in any
form of anti-coagulant
[0073] In view of the above, the microfluidic coagulation device
100 presented here is simple to operate, automated, and
multifunctional in that it also can be used to analyze platelet
aggregation in combination with the relations disclosed herein (Eq.
(1)-Eq. (4)) and can provide an enhanced, real-time quantitative
assay for monitoring whole blood thrombogenicity, such as a
patient's bedside, in a clinical laboratory, or even as a
home-care-based assessment tool.
[0074] In accord with another aspect of the present concepts, a
method (performed in vivo or in vitro) of assessing an effect of a
modifier on blood coagulation (e.g., determination of heparin
sensitivity by varying heparin levels, such as was shown in FIGS.
4A-4D, etc.) includes the acts of driving a first portion of a
blood sample at a constant flow rate through a first plurality of
microchannels 110 formed in a microfluidic device 100 substrate and
measuring a pressure, or a variable correlated with pressure, in or
across at least one of the first plurality of microchannels while
the first portion of the blood sample is moved through the first
plurality of microchannels at the constant flow rate. The method
further includes the acts of determining a first pressure value at
an initiation of flow of the first portion of the blood sample and
determining a first time at which a second pressure value of the
first portion of the blood sample is determined to be about twice
the determined first pressure value of the first portion of the
blood sample. The method further includes the acts of determining a
second time at which a third pressure value of the first portion of
the blood sample is determined to be about (1+e) times the
determined first pressure value of the first portion of the blood
sample and establishing a coagulation model predictive of channel
occlusion for the first portion of the blood sample using the first
time and the second time, for the first portion of the blood
sample, in accord with the relation in Eq. (1), wherein T.sub.pf is
the second time and T.sub.pg is the first time. The method further
includes the acts of driving a second portion of the blood sample
at a constant flow rate through a second plurality of microchannels
formed in the microfluidic device substrate or another microfluidic
device substrate and adding a modifier to one of the second portion
of the blood sample or the second plurality of microchannels. The
method further includes the acts of measuring a pressure, or a
variable correlated with pressure, in at least one of the second
plurality of microchannels while the second portion of the blood
sample is moved through the second plurality of microchannels at
the constant flow rate, determining a first pressure value at an
initiation of flow of the second portion of the blood sample, and
determining a first time at which a second pressure value of the
second portion of the blood sample is determined to be about twice
the determined first pressure value of the second portion of the
blood sample. The method further includes the acts of determining a
second time at which a third pressure value of the second portion
of the blood sample is determined to be about (1+e) times the
determined first pressure value of the second portion of the blood
sample and establishing a coagulation model predictive of channel
occlusion for the second portion of the blood sample using the
first time and the second time, for the second portion of the blood
sample, in accord with the relation of Eq. (1), wherein T.sub.pf is
the second time and T.sub.pg is the first time. The method further
includes the acts of comparing the coagulation model predictive of
channel occlusion for the first portion of the blood sample to the
coagulation model predictive of channel occlusion for the second
portion of the blood sample to determine an effect of the modifier
(e.g., an effect on overall coagulation time, an effect on a
particular constituent element to coagulation, etc.).
[0075] In still another aspect of the present concepts, a method
(performed in vivo or in vitro) of assessing an effect of a
modifier on blood coagulation (e.g., determination of heparin
sensitivity by varying heparin levels, such as was shown in FIGS.
4A-4D, etc.) includes the acts of driving a first portion of a
blood sample at a constant pressure through a first plurality of
microchannels 110 formed in a microfluidic device 100 substrate and
measuring a flow rate, or a variable correlated with flow rate, in
or across at least one of the first plurality of microchannels
while the first portion of the blood sample is moved through the
first plurality of microchannels at the constant pressure. The
method further includes the acts of determining a first flow rate
value at an initiation of flow of the first portion of the blood
sample, determining a first time at which a second flow rate value
of the first portion of the blood sample is determined to be about
twice the determined first flow rate value, and determining a
second time at which a third flow rate value of the first portion
of the blood sample is determined to be about (1+e) times the
determined first flow rate value. The method further includes the
act of establishing a first coagulation model predictive of channel
occlusion for the first portion of the blood sample using the first
time and the second time in accord with the relation of Eq. (2),
wherein T.sub.qf is the second time and T.sub.qg is the first time.
The method further includes the act of driving a second portion of
the blood sample at a constant pressure through a second plurality
of microchannels formed in the microfluidic device substrate or
another microfluidic device substrate and adding a modifier to one
of the second portion of the blood sample or the second plurality
of microchannels.
[0076] The modifier may comprise, by way of example, an
anti-coagulant (e.g., heparin, a low molecular weight heparin, a
direct factor inhibitor, a direct thrombin inhibitor, an
antithrombin protein, rivorxaban, apixaban, debigatran, a coumarin,
hirudin, lepirudin, bivalirudin, argatroban, dabigatran,
batroxobin, hementin, etc.), a food supplement derivative, an
anti-platelet drug (e.g., an irreversible cyclooxygenase inhibitor,
an adenosine diphosphate (ADP) receptor inhibitor, a
phosphodiesterase inhibitor, a glycoprotein IIb/IIIa inhibitor, an
adenosine reuptake inhibitor, a thromboxane inhibitor, etc.), or a
thrombolytic drug (tissue plasminogen activator (tPA),
streptokinase, urokinase, etc.). In accord with the present
concepts, the modifier can be any substance, or combination of
substances, that may affect one or more aspects of the coagulation
cascade or that does, in fact, affect one or more aspects of the
coagulation cascade. In accord with the present concepts, the
modifier can also comprise a modification of removing any
substance, or combination of substances, that may affect one or
more aspects of the coagulation cascade or that does, in fact,
affect one or more aspects of the coagulation cascade. In accord
with yet other aspects of the present concepts, the modifier can
also comprise both an addition of one or more substances (e.g., a
drug under test) that may affect one or more aspects of the
coagulation cascade or that does, in fact, affect one or more
aspects of the coagulation cascade and a removal of another one or
more substances (e.g., a cell, a cellular component, a protein,
etc.) that may affect one or more aspects of the coagulation
cascade or that does, in fact, affect one or more aspects of the
coagulation cascade (e.g., removing platelets to focus on fibrin in
a test of a drug potentially affecting fibrin, etc.).
[0077] The method further includes the acts of measuring a flow
rate, or a variable correlated with flow rate, in or across at
least one of the second plurality of microchannels while the second
portion of the blood sample is moved through the second plurality
of microchannels at the constant pressure and determining a first
flow rate value at an initiation of flow of the second portion of
the blood sample. The method further includes the acts of
determining a first time at which a second flow rate value of the
second portion of the blood sample is determined to be about twice
the determined first flow rate value of the second portion of the
blood sample and determining a second time at which a third flow
rate value of the second portion of the blood sample is determined
to be about (1+e) times the determined first flow rate value of the
second portion of the blood sample. The method further includes the
act of establishing a second coagulation model predictive of
channel occlusion for the second portion of the blood sample using
the first time and the second time, for the second portion of the
blood sample, in accord with the relation of Eq. (2), wherein
T.sub.qf is the second time and T.sub.qg is the first time. The
method further includes the act of comparing the coagulation model
predictive of channel occlusion for the first portion of the blood
sample to the coagulation model predictive of channel occlusion for
the second portion of the blood sample to determine an effect of
the modifier (e.g., an effect on overall coagulation time, an
effect on a particular constituent element to coagulation,
etc.).
[0078] More generally, the microfluidic coagulation device 100
comprises at least one substrate defining a plurality of
microchannels 110 (e.g., 2 microchannels, 3 microchannels, etc.).
The microchannels 110 are optionally arranged in parallel, and may
be formed in two or three dimensions. By varying the width and
height of each microchannel 110, it may comprise a cross-sectional
surface area in the range of 125 .mu.m.sup.2-1.75 mm.sup.2, which
may be uniform amongst the microchannels, or which may vary between
one or more of the microchannels or which may vary even along the
same microchannel. The sudden expansions to induce shear gradients
(flow acceleration and deceleration) can also be altered
accordingly. Moreover, a length, shape, surface treatment and/or
path of each microchannel 110 need not be uniform and a length,
shape, surface treatment and/or path of one or more microchannels
may differ from that of one or more other microchannels. By way of
example, one of the plurality of microchannels has a
cross-sectional geometry that is 75 .mu.m in height and 200 .mu.m
in width, whereas another one of the plurality of microchannels (or
possibly a different portion of the same microchannel) has a
cross-sectional geometry that is 150 .mu.m in height and 400 .mu.m
in width. Thus, in accord with one aspect of the microfluidic
device 100, at least one of the plurality of microchannels has a
first cross-sectional area and at least one of the plurality of
microchannels has a second cross-sectional area different than the
first cross-sectional area. As another example, in another aspect
of the microfluidic coagulation device 100, at least one of the
plurality of microchannels has a first surface treatment (e.g., a
naturally occurring or synthetic reagent, collagen, a thrombus
formation-inducing material, a thrombus formation-inhibiting
material, cells, endothelial cells, smooth muscle cells, segmented
polyurethane, polyvinyl chloride, or polymethyl-methacrylate, etc.)
and at least one of the plurality of microchannels has a second
surface treatment different than the first surface treatment.
[0079] In accord with the disclosed microfluidic device 100, a
first port is provided at a first end portion of the substrate
(e.g., a proximal or distal end of the substrate), the first port
connecting to an inlet end of the plurality of microchannels, or a
channel or microchannel leading to the first end of the plurality
of microchannels. A second port is provided at a second end portion
of the substrate (e.g., the other of the proximal or distal end of
the substrate), the second port connecting to outlet ends of the
plurality of microchannels, or channel or microchannel leading to
the second end of the plurality of microchannels. In accord with
the system depicted in FIG. 1, a pump (e.g., a syringe pump) is
attached to a port (e.g., a first port 120a) and configured to
apply a differential pressure across the first port to drive a
blood sample across the plurality of microchannels 110 at a
substantially constant flow rate or constant pressure. In the
experimental set-up, the tube 125 connecting the pump to the
microfluidic device 100 was bonded to the microfluidic device 100.
In other configurations, the port and tube 125 may advantageously
comprise quick-lock connectors such as, but not limited to,
push-to-connect components or a Luer-Lock.RTM. connection
fitting.
[0080] A first sensing device is configured to determine a pressure
value in, or relating to, a pressure across the plurality of
microchannels. For example, this first sensing device may comprise
one or more pressure sensing devices. In another example, the first
sensing device comprises a sensor configured to determine a value
relating to a pressure across the plurality of microchannels (e.g.,
amperage of pump). In yet another example, the pressure sensing
device senses the negative pressure while the pump delivers vacuum
pressure.
[0081] As previously noted, FIG. 10 shows an analytical model for
quantitative assessment of whole blood coagulation on a
microfluidic device 100 operating in infusion pump mode or pressure
pump mode in accord with at least some aspects of the present
concepts wherein, in infusion mode, the pressure grows
exponentially and wherein, in pressure mode, the decay in flow
follows a sigmoid trend, with the clotting times able to be
extracted by fitting the equations of the analytical model to these
measurements respectively. Also shown by a vertical dashed line at
approximately 30 minutes are the times T.sub.pg,T.sub.qg, which
represent physiologically the growth phase of clotting under
constant flow and constant pressure, respectively. Also shown by a
vertical dashed line at approximately 50 minutes are the times
T.sub.pf, T.sub.qf, which represent physiologically, the saturation
phase of clotting under constant flow and constant pressure,
respectively. Superimposed on FIG. 10 are representations of
cross-sections of the microchannels 110 at T=0 (no clotting),
T=T.sub.pg,T.sub.qg (growth phase, showing partial occlusion of the
cross-section), and T=T.sub.pf, T.sub.qf (saturation phase, at
least substantially complete occlusion).
[0082] FIG. 11 shows whole blood, drawn in sodium citrate, perfused
through the microfluidic device 100 of FIG. 1, and .DELTA.P-Q
response curve is plotted to estimate .DELTA.P(0) and Q(0) in
infusion mode and pressure mode of operation respectively in accord
with at least some aspects of the present concepts.
[0083] FIG. 12 shows an analytical model to predict the clotting
time of whole blood in the coagulation monitoring microfluidic
device as a function of shear rate/stress in accord with at least
some aspects of the present concepts.
[0084] The microfluidic coagulation device 100 shown in FIG. 1
advantageously comprises an attendant computer system, comprising a
controller including one or more processors, a bus or other
communication mechanism coupled to the one or more processors for
communicating information, and a main memory (e.g., RAM) and/or
other dynamic storage device, coupled to the bus for storing
information and instructions to be executed by processor. This
computer system is still further advantageously integrated together
with the pump and control systems to enable computer control of the
pump operation and sensor data collection. The main memory also may
be used for storing temporary variables (e.g., pressure, flow rate,
time, etc.) or other intermediate information during execution of
instructions to be executed by the controller. Such computer system
also includes ROM or other static storage device coupled to the bus
for storing static information and instructions for processor. A
physical computer-readable storage device, such as a solid-state
memory device, is provided and coupled to the bus for storing
information. The computer system is also coupled via the bus to one
or more display devices (e.g., flat screen display, touch screen,
etc.), one or more input devices (keypad, keys, mouse, etc.). In
accord with the disclosed methods, in at least some aspects, the
methods are implemented utilizing the computer system in response
to controller executing one or more sequences of one or more
instructions contained in a physical memory device attached to the
bus, such as the main memory. Execution of the sequences of
instructions causes the controller to perform at least some of the
process steps described herein. By way of example, the memory
device(s) bear instructions configured to cause the controller to
determine, in combination with inputs from the sensing device(s)
and a timer, a first pressure value at an initiation of flow, a
first time at which a second pressure value is determined to be
about twice the determined first pressure value, and a second time
at which a third pressure value is about (1+e) times the determined
first pressure value. The memory device(s) also bear instructions
configured to cause the controller to determine to establish a
patient coagulation model predictive of channel occlusion in accord
with the relation of Equation (1), above. Similarly, in another
aspect, the memory device(s) bear instructions configured to cause
the controller to determine, in combination with inputs from the
sensing device(s) and a timer, a first flow rate value at a first
time corresponding to an initiation of flow, a second time at which
a second flow rate value is determined to be about half the
determined first flow rate value, a third time at which a third
flow rate value is determined to be about (1+e) times lesser than
the determined first flow rate value, and a patient coagulation
model predictive of channel occlusion governed by the relation of
Equation (2), above.
[0085] The term "computer-readable medium" as used herein refers to
any physical medium that participates in providing instructions to
processor(s) for execution (e.g., non-volatile media, volatile
media, magnetic media, optical media, solid state media, etc.). The
computer system utilized in combination with the microfluidic
device 100 also advantageously, but optionally, includes a
communication interface coupled to the bus, such communication
interface providing a two-way data communication coupling to a
network link (e.g., an integrated services digital network (ISDN)
card, modem, local area network (LAN) card, wireless link, etc.).
The network link provides data communication through one or more
networks to other data devices (e.g., the network link may provide
a connection through local network to a host computer or to data
equipment operated by an Internet Service Provider (ISP)) and the
computer system is configured to send and receive data through the
network(s), network link(s), and communication interface(s).
[0086] It is further noted that the present concepts enable a given
patient therapy (e.g., a self-infusions, home care, clinic care,
remote health monitoring, etc.) to be modified in real-time. A
physician, medical care provider, nurse (or potentially a trained
patient) is able to track progress of therapy with the disclosed
microfluidic coagulation device 100 and developed equations
(1)-(4). For example, while a patient is receiving an infusion
(e.g., BeneFIX.RTM., Rixubis, etc.) to provide a therapeutic effect
as to a particular malady (e.g., Hemophilia B, etc.), the physician
or nurse (or patient) can track the progress of the therapy during
the infusion to determine efficacy for that particular patient at
that particular time, rather than relying on less precise
population estimates. Particularly for expensive treatment
protocols (e.g., Factor IX replacement, etc.), the real-time
coagulation assessment provided in accord with the present concepts
potentially enables treatment to be stopped when an actual,
appropriate hemostatic balance has been achieved, rather than
relying on gross estimates (or over-estimates) for an infusion
dosage required, thus reducing both the cost of treatment (e.g., it
could be determined that 500iU was not needed and that 250iU was
therapeutically sufficient) and the risk of potentially attendant
side effects. Continuing with the example of a patient having
Factor IX deficiency, real-time changes in the patient's Factor-IX
levels (e.g., responsive to diet, ingested supplement, medicine,
etc.) may yield a generalized dosing requirement insufficient or,
conversely, if a patient's generalized dosing requirement is
determined in a clinic at a time at which the patient's Factor-IX
levels are suppressed from a typical baseline (e.g., responsive to
diet, ingested supplement, medicine, etc.), the prescribing dosing
may be more than would be required to achieve the desired
therapeutic benefit. Thus, the present concepts permit tailoring of
a therapeutic treatment, in real-time, to the specific patient
(e.g., human or animal).
[0087] These present concepts present many potential applications.
In general, coagulation monitors of various types may be used in
the diagnosis of thrombogenic disorders (e.g., atherosclerosis,
deep venous thrombosis, bleeding disorders, etc.), direct
intravascular coagulation (e.g., sepsis, sickle cell disease,
trauma, etc.), blood transfusion, hemofiltration, cardiac therapy
(e.g., stents, angioplasty, etc.) and monitoring the dosage of
anti-coagulation therapy for any of the condition described above.
In addition, coagulation monitoring devices are used in drug
development research and assays to determine platelet function,
etc. The microfluidic device 100 disclosed herein may be used in
any of the above applications or settings. Moreover, the disclosed
microfluidic device 100 and systems and methods relating thereto,
can be performed in such applications or settings at either
constant flow or constant pressure driven flow, or potentially both
constant flow and constant pressure driven flow.
[0088] By way of example, the disclosed microfluidic device 100 and
systems and methods relating thereto may be advantageously utilized
in the applications of (1) anti-coagulation therapy, (2)
anti-platelet therapy, (3) platelet function tests, (4)
determination of surface thrombogenicity, and (5) shear stress
response. As to anti-coagulation therapy, the present concepts may
be applied, for example, to monitor the dosage of and efficacy of
both traditional anti-coagulants, such as heparin and warfarin, and
new or developmental drugs such as, but not limited to, dabigatran,
lepirudin, apixaban, and/or rivaroxaban. As to anti-platelet
therapy, the present concepts may be applied, for example, to
monitor the dosage of and/or efficacy of anti-platelet drugs such
as, but not limited to, aspirin, rofecoxib, valdecoxib,
clopidogrel, prasugrel and/or abciximab. As noted above, the
microfluidic device 100 and associated system and methods permit
dosage to be adjusted, and optimized, in real-time during therapy
(e.g., by integrating the device and control system to
extra-corporeal treatments, by utilizing the device and control
system in a bed-side testing unit, etc.) or substantially
contemporaneously therewith (e.g., tests can be undertaken at short
time-intervals).
[0089] As to the platelet function tests, the disclosed
microfluidic device 100 and systems and methods relating thereto
are able to unravel platelet activation and aggregation biology and
biophysics. The platelet integrins can bind collagen, laminin, and
fibrinogen. Platelet activation is also associated with release of
ADP and serotonin, synthesis of thromboxane, and exposure of
phosphatidylserine, which facilitates thrombin generation.
Micropatterned surfaces of hemostatically active proteins such as
fibrinogen, collagen (I-VIII), vWF, and lipidated tissue factor can
be selectively, serially or parallelly coated on partial or full
surface of the device and a multiplexed assay can be carried out.
This may also include endothelial cells. Platelet aggregation can
also be tested by adding variety of platelet agonists to the blood
sample that may include ADP, epinephrine, collagen, arachidonic
acid, thrombin , ristocetin etcetera. The surface pattern and/or
agonist used could help determine the medication of a patient who
is taking some form of anti-coagulation drug depending upon
condition.
[0090] The disclosed microfluidic device 100 and systems and
methods relating thereto are further able to be used for
determination of surface thrombogenicity. Platelets may interact
with naturally occurring material (endothelial cells, collagen) or
synthetic material (e.g., polyethylene glycol (PEG), PEO, POE,
poly(1,8-octanediol citrate) (POC), etc.) that could determine
hemocompatibility of biomedical devices. The microfluidic device
100 can be coated with a variety of such natural and/or or
synthetic materials and hemocompatibility of these materials can be
determined with this device.
[0091] Further, in view of the above, it is to be understood that
one or more of the microfluidic device 100 microchannels 110, or
portion(s) thereof, can be coated with a variety of such natural
and/or or synthetic materials and hemocompatibility of these
materials can be determined with this device. For example, one or
more microchannels 110 of the microfluidic device 100, or
portion(s) thereof, can be coated with a first naturally occurring
material (e.g., Type 1 collagen), one or more microchannels of the
microfluidic device, or portion(s) thereof, can be coated with a
second naturally occurring material (e.g., Type 2 collagen), and
one or more microchannels of the microfluidic device, or portion(s)
thereof, can be coated with a third naturally occurring material
(e.g., Collagen alpha-1 (III) chain), and one or more microchannels
of the microfluidic device, or portion(s) thereof, can be coated
with a synthetic material (e.g., PEG). As another example, one
microchannel 110, or portion(s) thereof, is coated with collagen,
another microchannel, or portion(s) thereof, is coated with
epinephrine and yet another microchannel, or portion(s) thereof, is
coated with Thromboxane A2 (TXA2) to enhance assay specificity and
sensitivity. In accord with at least some aspects of the present
concepts, one or more microchannels 110 can be used as controls,
providing known or standardized results to which behavior of one or
more other microchannels 110 can be readily compared. In yet other
aspects, at least some of the microchannels of the microfluidic
device 100 are adapted to provide diagnostic tools, such as to
detect the D-dimer antigen as a marker to rule out the presence of
venous thromboembolism (e.g., deep vein thrombosis (DVT) and/or
pulmonary embolism (PE)), or research tools, such as to study
extracellular matrix (ECM) induced clot formation.
[0092] The microfluidic device 100 may accordingly comprise, for
example, a plurality of microchannels 110 having one or more
different coatings and/or surface treatments that may comprise one
or more natural materials, one or more synthetic materials, or a
combination of one or more natural materials (e.g., cells,
proteins, molecules, enzymes, receptors, etc.) and one or more
synthetic materials.
[0093] Additionally, the disclosed microfluidic device 100 and
systems and methods relating thereto are able to be used for
determination of shear stress response. Coagulation is a function
of shear stress and the disclosed microfluidic device 100 can allow
a relevant shear stress to be applied to quantitatively assess
thrombogenicity in the range 75-2500 sec.sup.-1.
[0094] Recent mechanistic studies have shown that thrombus
formation and platelet aggregation at the site of vascular injury
or atherosclerotic lesions, in vivo, are caused by changes in the
fluid dynamics of blood flow. More specifically, independent of
soluble clotting agonists, thrombus formation and platelet
aggregation is enhanced due to shear gradients (rate of change of
shear stress) arising from acceleration and deceleration of flow at
stenotic regions and clotting is most pronounced post-stenosis,
where the flow decelerates. However, the agonists, such as collagen
or von Willebrand factor (vWf), may also contribute in stabilizing
the clot. FIG. 13A shows another example of a microfluidic device
100, in accord with at least some aspects of the present concepts,
comprising a microchannel 110 geometry that allows the fluid to
pass through a converging zone 500 (flow acceleration; prestenosis)
near the inlet into multiple lanes made of channels of constant
width 520 that have curves and straight sections. The fluid then
exits through a diverging section 540 (flow deceleration;
post-stenosis) into a common outlet.
[0095] As shown in FIGS. 13A-13C, finite element computational
modeling of non-Newtonian blood flow, using COMSOL
Multiphysics.RTM. software, predicts that the wall shear rate
increases dramatically at the converging section 500 (see reference
numeral 600 FIG. 13A, Wall Shear Rate, .gamma.[sec.sup.-1] vs.
Distance, x [mm], and the corresponding exploded view of reference
numeral 600 in FIG. 13B). The computational modeling also predicts
that the wall shear rate will remain steady in the constant-width
section 520 and decrease dramatically at the diverging section 540.
In the computational model, the inlet boundary condition imposed is
P=0 (no pressure) and the outlet boundary condition is of a
specified normal velocity (no flow across the channel and only
along the longitudinal/vertical direction).
[0096] In the Wall Shear Rate, .gamma.[sec.sup.-1] vs. Distance, x
[mm] plot at the bottom of FIG. 13A and in FIG. 13B, plots are
shown (from top to bottom) for values of normal velocity, u=0.5,
0.2, 0.1, 0.05, 0.025 and 0.01, [mm/sec] respectively
[0097] Therefore, in the embodiment of microfluidic device 100
shown in FIG. 13A, the shear rate gradients that are created depend
on the mean wall shear that is applied and controlled from an
external blood pump. However, the geometry can be altered to have
any desirable hemodynamic environment that mimics or exacerbates
the functional pathophysiology of atherosclerosis for the purpose
of improving blood based analytical devices.
[0098] For the microfluidic device 100 of FIG. 13A, it is shown in
FIG. 14 that fibrin formation 610 and platelet adhesion 620 are
elevated at the converging (upstream) and diverging (downstream)
sections 500, 540 of the microfluidic device, where the shear
gradient is very high (as shown in FIG. 13C), as compared to the
remaining constant-width section 520 of the device. Interestingly,
as shown in FIG. 13C and FIG. 14, blood clotting is maximized at
the diverging (downstream) section 540, which mimics the
post-stenotic geometry of atherosclerosis. Therefore, the disclosed
microfluidic device 100 allows blood clotting to occur and to be
easily visualized in the presence of physiological or pathological
shear and shear gradients, inside microchannels 110 that mimic the
size of human blood vessels.
[0099] As noted above, the microfluidic device 100 can be made of a
variety of materials that can be prothrombogenic or
anti-thrombogenic. For example, the thrombus formation inside a
microfluidic device 100 made of PDMS with no surface alteration of
the microchannels 110, when activated by shear gradient alone, such
that the maximum gradient achieved is 3.5 times the mean wall
shear, results in clotting time in the range of 15-60 minutes
depending upon parameters such as, concentration of anticoagulant,
applied shear, etc. However, when the microfluidic device 100
microchannels 110 are coated with human collagen I at a
concentration of 100 .mu.g/ml, it was found that the clotting time
can be reduced to 2-20 minutes and therefore, allowing this tool
for a more rapid analysis, when desired, as is shown in FIG.
15.
[0100] The thrombus monitoring device can be operated, ex vivo, by
directly attaching it to catheters or extra-corporeal devices and
thus enabling analysis if native blood not drawn in any form of
anti-coagulant.
[0101] It is known that equipment used to draw blood, primarily
including some type of anticoagulation tube (EDTA, heparin, PPACK
etc), changes the blood chemistry that may impact many in vitro
hemoanalytical assays, including clotting time tests. Aspects of
the disclosed microfluidic device 100, such as that depicted in
FIGS. 13A and 14, which require a very small amount of blood and
relatively simple instrumentation downstream of the device, can
potentially be attached directly to a patient's blood vessel (e.g.,
via a standard catheter) or integrated to an extra-corporeal device
(e.g., a cardiac pump, ECMO device, dialysis equipment, etc.), thus
minimizing the impact of anticoagulation tube and other
pre-analytical variables that alter blood function. Thus, native
whole blood from a patient can be passed directly to the
microfluidic device 100 without need for any external pump,
intermediary storage, or treatment (e.g., anti-thrombogenic
coatings are not needed and are advantageously omitted).
[0102] In accord with the above concepts, the clotting
characteristics of a blood sample in the microfluidic device 100
can be tailored by selection of the microchannel 110 geometry
(e.g., to alter a shear stress gradient, etc.) and/or selection of
optional agonists. As one example, the microfluidic device 100 of
FIG. 13A comprises a plurality of microchannels (e.g., more than
one microchannel), each of the plurality of microchannels defining
a proximal first end (comprising an inlet portion), a medial
portion, and a distal second end (comprising an outlet portion).
Each of the microchannels 110 is the example of FIG. 13A comprises
a converging portion (e.g., preferably comprising a gently
converging cross-sectional area over a first length, but
potentially comprising a step-decrease) where a cross-sectional
area of the microchannel decreases from a first cross-sectional
area to a smaller second cross-sectional area. Over the medial
portion, which may occupy a significant portion of a length of the
microchannel 110, a substantially constant cross-sectional area is
maintained. A diverging portion (e.g., preferably comprising a
gently diverging cross-sectional area over a third length, but
potentially comprising a step-increase) is also provided where the
cross-sectional area of the microchannel increases from the second
cross-sectional area to a larger third cross-sectional area. The
third cross-sectional area may be the same as or different from the
first cross-sectional area. As with other embodiments of the
microfluidic device described herein, the plurality of
microchannels 110 may advantageously comprise a first set of one or
more microchannels having a first geometry over at least a portion
of its length and a second set of one or more microchannels having
a second geometry over at least a portion of its length. These
first and second sets of one or more microchannels may further
optionally comprise the same agonist and/or surface treatments or
different agonists and/or surface treatments, as described
elsewhere herein.
[0103] In other aspects of the present concepts, at least some of
the plurality of microchannels 110 may omit either the converging
portion or the diverging portion, with the wall shear gradient
being selected in the respective one of the converging portion or
the diverging portion, or collectively the whole of the
microchannel, to achieve a desired hemodynamic environment.
Further, although examples are provided herein with converging
portions in inlet regions of a microchannel and diverging portions
in outlet regions of the microchannels, such converging portions
and diverging portions are not limited to the periphery of the
microchannels and may be disposed anywhere within the microchannels
(e.g., in a middle portion of the microchannels) and may comprise
any number of such converging portions and diverging portions
(e.g., two sets or three sets of converging portions and diverging
portions).
[0104] As disclosed herein, substantially constant flow and
substantially constant mean that the flow or pressure,
respectively, may vary within a range about an average or mean flow
or pressure value, such as by +/-10%, +/-5%, +/-2%, or +/-1%
depending upon the specifications of instrumentation/equipment used
to set the substantially constant flow or pressure.
[0105] Each of the disclosed embodiments and obvious variations
thereof are contemplated as falling within the spirit and scope of
the claimed invention, aspects of which embodiments are set forth
in the following claims.
* * * * *