U.S. patent application number 15/916550 was filed with the patent office on 2018-07-12 for systems, devices, and methods for measuring whole blood hematocrit based on initial fill velocity.
The applicant listed for this patent is LifeScan, Inc.. Invention is credited to Ronald C. Chatelier, Alastair M. Hodges, Linda Raineri, Dennis Rylatt.
Application Number | 20180195994 15/916550 |
Document ID | / |
Family ID | 43779499 |
Filed Date | 2018-07-12 |
United States Patent
Application |
20180195994 |
Kind Code |
A1 |
Chatelier; Ronald C. ; et
al. |
July 12, 2018 |
SYSTEMS, DEVICES, AND METHODS FOR MEASURING WHOLE BLOOD HEMATOCRIT
BASED ON INITIAL FILL VELOCITY
Abstract
Methods for determining the hematocrit of a blood sample, and
devices and systems used in conjunction with the same. The
hematocrit value can be determined on its own, and further, it can
be further used to determine a concentration of an analyte in a
sample. In one exemplary embodiment of a method for determining the
hematocrit value in a blood sample, a volume of blood is provided
in a sample analyzing device having a working and a counter
electrode. An electric potential is applied between the electrodes
and an initial fill velocity of the sample into the device is
calculated. The hematocrit of the blood, as well as a concentration
of an analyte in view of the initial fill velocity can then be
determined. Systems and devices that take advantage of the use of
an initial fill velocity to determine hematocrit levels and make
analyte concentration determinations are also provided.
Inventors: |
Chatelier; Ronald C.;
(Bayswater, AU) ; Rylatt; Dennis; (Wheelers Hill,
AU) ; Raineri; Linda; (Prahran, AU) ; Hodges;
Alastair M.; (Blackburn, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LifeScan, Inc. |
Wayne |
PA |
US |
|
|
Family ID: |
43779499 |
Appl. No.: |
15/916550 |
Filed: |
March 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14511235 |
Oct 10, 2014 |
9927388 |
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15916550 |
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|
12649509 |
Dec 30, 2009 |
8877034 |
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14511235 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 27/3273 20130101;
G01N 2333/4737 20130101; G01N 27/3274 20130101; G01N 33/49
20130101; G01N 27/3272 20130101; G01N 33/80 20130101; G01N 27/48
20130101 |
International
Class: |
G01N 27/327 20060101
G01N027/327; G01N 33/49 20060101 G01N033/49; G01N 33/80 20060101
G01N033/80; G01N 27/48 20060101 G01N027/48 |
Claims
1. A method for determining a concentration of an analyte in a
sample, the method comprising: providing a sample including an
analyte to a sample analyzing device having a working electrode and
a counter electrode; applying an electric potential between the
working electrode and the counter electrode; determining an initial
fill velocity of the sample; and calculating a concentration of the
analyte in view of the initial fill velocity.
2. The method of claim 1, wherein determining an initial fill
velocity further comprises: measuring an initial current after
applying the electric potential; determining a level of hematocrit
in the sample; and reversing the electric potential between the
working electrode and the counter electrode.
3. The method of claim 2, wherein calculating the concentration of
the analyte further comprises computing the concentration based on
the determined level of hematocrit.
4. The method of claim 3, further comprising measuring a change in
current over a period of time following reversing the electric
potential, wherein calculating a concentration of the analyte
further comprises calculating the concentration of the analyte in
view of the change in current over the period of time.
5. The method of claim 3, wherein the sample comprises whole blood,
the method further comprising at least one of measuring a
temperature of the whole blood or inferring a temperature of the
whole blood.
6. The method of claim 1, wherein determining an initial fill
velocity further comprises determining a rate of change in an
optical signal to calculate the initial fill velocity.
7. The method of claim 1, wherein determining an initial fill
velocity further comprises determining an initial current flow to
determine the initial fill velocity.
8. The method of claim 7, wherein determining an initial current
flow further comprises: performing current measurements
approximately every 10 milliseconds for at least approximately 50
milliseconds; and calculating an average current based on the
current measurements.
9. The method of claim 1, wherein determining an initial fill
velocity further comprises determining an initial fill velocity
directly after the sample enters a capillary space of the sample
analyzing device.
10. The method of claim 1, wherein determining an initial fill
velocity further comprises determining an initial fill velocity
after the sample crosses into a region of a capillary space of the
sample analyzing device where a detection signal is generated.
11. The method of claim 1, wherein the sample analyzing device
comprises an immunosensor.
12. The method of claim 3, further comprising measuring a change in
current over a period of time after reversing the electric
potential, wherein calculating a concentration of the analyte
further comprises calculating the concentration of the analyte in
view of the change in current over the period of time.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Ser.
No. 14/511,235, filed on Oct. 10, 2014, which is a divisional
application of U.S. Ser. No. 12/649,509 (now U.S. Pat. No.
8,877,034), issued on Nov. 4, 2014. The entire contents of each
application is incorporated herein by reference.
FIELD
[0002] The present disclosure relates to determining a
concentration of an analyte in a sample, and more particularly
relates to making a more accurate determination of the
concentration based on an initial fill velocity of the sample.
BACKGROUND
[0003] Analyte detection in physiological fluids, e.g. blood or
blood derived products, is of ever increasing importance to today's
society. Analyte detection assays find use in a variety of
applications, including clinical laboratory testing, home testing,
etc., where the results of such testing play a prominent role in
diagnosis and management in a variety of disease conditions.
Analytes of interest include glucose for diabetes management,
cholesterol, and the like. In response to this growing importance
of analyte detection, a variety of analyte detection protocols and
devices for both clinical and home use have been developed. Some of
these devices include electrochemical cells, electrochemical
sensors, hemoglobin sensors, antioxidant sensors, biosensors, and
immunosensors.
[0004] One characteristic of blood that can affect analyte
detection is the hematocrit. Levels of hematocrit can be vastly
different amongst various people. By way of non-limiting example, a
person suffering from anemia may have a hematocrit level of
approximately 20% while a neonate may have a hematocrit level of
approximately 65%. Even samples taken from the same individual over
a period of time can have different hematocrit levels. Further,
because high hematocrit can also increase the viscosity of blood,
and viscosity can in turn affect other parameters associated with
analyte detection, accounting for the effect of hematocrit on a
sample can be important in making accurate analyte concentration
determinations.
[0005] One way in which varying levels of hematocrit in a blood
sample have been accounted for is by separating the plasma from the
blood and then recalculating the concentration of the antigen with
respect to the adjusted plasma volume. Separation has been
achieved, for example, by performing a centrifugation step. Other
ways in which the varying levels of hematocrit in a blood sample
have been accounted for include using an average hematocrit in a
calculation or measuring a hematocrit in a separate step and then
calculating the concentration of the antigen with respect to the
plasma value. These methods, however, are believed to be
undesirable, at least because they involve unwanted sample
handling, take additional time, and lead to substantial errors in
the final determinations. Further, temperatures in environments
where samples are analyzed can also have a negative impact on the
accuracy of analyte concentration determination.
[0006] Accordingly, it would be desirable to develop a way to
obtain more accurate analyte concentration measurements that
account for a wide spectrum of hematocrit levels and temperatures.
It would also be desirable to develop a way to determine hematocrit
levels quickly.
SUMMARY
[0007] Applicants have recognized that it would be desirable to
develop a way to obtain more accurate analyte concentration
measurements that account for a wide spectrum of hematocrit levels
and temperatures with little or none of the attendant issues noted
previously. Applicants have also recognized that it would also be
desirable to develop a way to determine hematocrit levels quickly.
Accordingly, systems, devices, and methods are generally provided
for determining a hematocrit value of a blood sample and for
determining a concentration of an analyte in a sample. In one
exemplary embodiment of a method for determining a hematocrit value
of a whole blood sample, the method includes providing a sample of
whole blood to a sample analyzing device having a capillary space,
measuring an initial fill velocity of the sample in at least a
portion of the capillary space, and determining a hematocrit value
of the sample from the initial fill velocity. Measuring the initial
fill velocity can include applying an electrical potential,
measuring an electrical current, and determining an initial current
flow. In one embodiment, current measurements are performed
approximately every 10 milliseconds for at least approximately 50
milliseconds and an average current based on the current
measurements is calculated. In another alternative embodiment,
measuring the initial fill velocity can include detecting an
optical signal. In one embodiment, measuring an initial fill
velocity occurs directly after the sample enters the capillary
space. In still another embodiment, measuring an initial fill
velocity occurs after the sample crosses into a region of the
capillary space of the sample analyzing device where a detection
signal is generated. A temperature of the sample can be measured or
inferred. The measured or inferred temperature can be used to
determine the hematocrit value of the sample. In one exemplary
embodiment, the sample analyzing device includes an
immunosensor.
[0008] In addition to measuring a hematocrit level, the method can
also be used to determine a concentration of an analyte in a
sample. For example, the method for determining a hematocrit value
can include calculating a concentration of the analyte in view of
the determined hematocrit value. This can be achieved, for example,
by applying an electric potential, measuring an initial current
after applying the electric potential, and reversing the electric
potential. A change in current over a period of time can be
measured following the reversal of the electric potential. The
measured change in current over a period of time can also be used
to calculate a concentration of the analyte. In one embodiment, a
temperature of the sample can either be measured or inferred. In
such an embodiment, a measured change in current over a period of
time and the temperature of the sample can be used to calculate a
concentration of the analyte.
[0009] In an exemplary embodiment of a method for determining a
concentration of an analyte in a sample, the method includes
providing a sample including an analyte to a sample analyzing
device having a working and a counter electrode, applying an
electric potential between the working and counter electrodes,
determining an initial fill velocity of the sample, and calculating
a concentration of the analyte in view of the initial fill
velocity. In one embodiment, the initial fill velocity can be
determined by determining a rate of change in an optical signal. In
another embodiment, the initial fill velocity can be determined by
determining an initial current flow. The initial current flow can
be determined, for example, by performing current measurements
approximately every 10 milliseconds for at least approximately 50
milliseconds, and then calculating an average current based on the
current measurements. In yet another embodiment, the initial fill
velocity can be determined by measuring an initial current after
applying the electric potential, determining a level of hematocrit
in the sample, and reversing the electric potential between the
working and counter electrodes. Further, the concentration of the
analyte can be computed based on the determined level of
hematocrit.
[0010] The method for determining a concentration of an analyte can
further include measuring a change in current over a period of
time, i.e., the slope m of a current versus time graph, following
the reversal of the electric potential. As a result, a
concentration of the analyte, C.sub.O, can be calculated in view of
the change in current over the period of time. For example, the
concentration of the analyte can be calculated using the following
equation:
C.sub.O=-3.5+0.866exp(y)
where
y = m ( 1 - 0.01 H ) 0.83 ##EQU00001##
and H is the level of hematocrit. The level of hematocrit H can be
determined by using the following equation:
H=97.6-1.7658|i.sub.i|
where |i.sub.i| is the absolute value of the initial current.
[0011] The sample analyzing device can be an immunosensor. The
analyte for which the concentration is being analyzed can be
C-reactive protein. The analyzed sample can be blood. In one
embodiment, the blood includes whole blood. The method can further
include measuring a temperature T of the whole blood, or
alternatively, measuring an ambient temperature and using it to
infer the temperature T of the blood. The method can also further
include measuring a change in current over a period of time, i.e.,
the slope m of a current versus time graph, following the reversal
of the electric potential. As a result, a concentration of the
analyte, C.sub.O, can be calculated in view of the change in
current over the period of time. For example, the concentration of
the analyte can be calculated using the following equation:
C.sub.O=-5.7+1.78exp(y')
where
y ' = y 1 + 0.068 ( T - 25 ) , y = m ( 1 - 0.01 H ) 1.55 ,
##EQU00002##
and H is the level of hematocrit. The level of hematocrit H can be
determined by the following equation:
H=77.1-2.1|i.sub.i|+0.75T
where |i.sub.i| is the absolute value of the initial current.
[0012] In one exemplary embodiment of an electrochemical system,
the system includes an immunosensor having lower and upper
electrodes, a meter configured to apply a potential between the
lower and upper electrodes of the immunosensor, and a control unit
configured to measure an initial fill velocity of a sample
introduced into the immunosensor. The control unit is further
configured to use the initial fill velocity to calculate at least
one of a value of hematocrit of the sample when the sample includes
blood and a concentration of an analyte in the sample. The system
can also include a heating element that is configured to heat at
least a portion of the immunosensor.
[0013] The immunosensor can include a first liquid reagent, a
second liquid reagent, and magnetic beads conjugated to an antigen.
In one embodiment, the first liquid reagent can include an antibody
conjugated to an enzyme in a buffer. The first liquid reagent can
be striped on the lower electrode and can be dried. The second
liquid reagent can include ferricyanide, a substrate for the
enzyme, and a mediator in a dilute acid solution. The second liquid
reagent can be striped on the lower electrode and can be dried. The
magnetic beads, on the other hand, can be striped on the upper
electrode and dried.
[0014] The immunosensor can also include a plurality of chambers, a
separator, a vent, and one or more sealing components. The
separator can be disposed between the lower and the upper
electrodes. The plurality of chambers can include a reaction
chamber, a detection chamber, and a fill chamber. The reaction
chamber can be formed in the separator and can have the first
reagent and the magnetic beads conjugated to the antigen disposed
therein. The detection chamber can also be formed in the separator
and can have the second reagent disposed therein. The fill chamber
can be formed at least partially in the separator and one of the
lower and upper electrodes, can be spaced a distance apart from the
detection chamber, and can overlap at least a portion of the
reaction chamber. The vent can be formed at least partially in each
of the separator, the lower electrode, and the upper electrode, can
be spaced a distance apart from the reaction chamber, and can
overlap at least a portion of the detection chamber. In one
embodiment, the one or more sealing components can be a first
sealing component and a second sealing component. The first sealing
component can have an incorporated anticoagulant coupled to one of
the lower and upper electrodes, can be disposed over the vent, and
can be configured to both form a wall of the fill chamber and seal
the vent. The second sealing component can be coupled to the other
of the lower and upper electrodes, can be disposed over the vent,
and can be configured to seal the vent. In one embodiment, the
first sealing component includes a hydrophilic adhesive tape.
[0015] In one embodiment, the control unit of the electrochemical
system can include an optical signal detector that is configured to
measure a rate of change in an optical signal to measure the
initial fill velocity of the sample. In another embodiment, the
control unit can include a current flow detector configured to
measure an initial current flow to measure the initial fill
velocity of the sample. In still another embodiment, the control
unit can be configured to measure the initial fill velocity of the
sample directly after the sample enters a capillary space of the
immunosensor. In yet another embodiment, the control unit can be
configured to measure the initial fill velocity after the sample
crosses into a region of a capillary space of the immunosensor
where a detection signal is generated. At least one of the control
unit, the immunosensor, and the meter can be configured to measure
a temperature of the sample or infer a temperature of the
sample.
[0016] The analyte for which the system calculates the
concentration can be C-reactive protein. The sample introduced into
the immunosensor can be blood. In one embodiment, the blood
includes whole blood.
[0017] The sample analyzing device can also be a number of other
analyzing devices, including, by way of non-limiting example,
electrochemical cells, electrochemical sensors, glucose sensors,
glucose meters, hemoglobin sensors, antioxidant sensors, and
biosensors. In one embodiment, the sample analyzing device includes
a glucose sensor. The glucose sensor can include an electrochemical
cell having a working electrode and a counter or counter/reference
electrode. The working electrode and the counter or
counter/reference electrode can be spaced apart by approximately
500 micrometers or less. In one embodiment, a spacing between the
electrodes is in the range of about 80 micrometers to about 200
micrometers. The spacing can be determined in order to achieve a
desired result, for example, substantially achieving a steady state
current in a desirable time. In one embodiment, a spacing between
the electrodes is selected such that the reaction products from a
counter electrode arrive at a working electrode.
[0018] The working and counter or counter/reference electrode can
have a variety of configurations. For example, the electrodes can
be facing each other, they can be substantially opposed to each
other, or they can have a side-by-side configuration in which the
electrodes are positioned approximately in the same plane. The
electrodes can have substantially the same corresponding area. The
electrodes can also be planar. In one embodiment, the
electrochemical cell includes a working electrode, a counter
electrode, and a separate reference electrode. In another
embodiment, the electrochemical cell can have two electrode pairs.
The electrode pairs can include any combination of working,
counter, counter/reference, and separate reference electrodes, but
in one exemplary embodiment, each pair includes a working electrode
and a counter or counter/reference electrode. In still another
embodiment, the electrochemical cell can have an effective cell
volume of about 1.5 microliters or less. The electrochemical cell
can alternatively be hollow.
[0019] A potential can be applied to the electrodes of the cells by
a number of different mechanisms, including, by way of non-limiting
example, a meter. The magnitude of the potential can depend on a
number of different factors, including, by way of non-limiting
example, the desired reaction of the sample within the cell. In one
embodiment, the magnitude of the potential can be selected such
that electro-oxidation of a reduced form or electro-reduction of an
oxidized form of a sample is substantially diffusion
controlled.
[0020] Samples can enter the cell by way of capillary action. A
control unit can be used to determine an initial velocity of the
sample entering the cell. In one embodiment, the control unit can
include an optical signal detector that is configured to measure a
rate of change in an optical signal to measure the initial fill
velocity of the sample. In another embodiment, the control unit can
include a current flow detector configured to measure an initial
current flow to measure the initial fill velocity of the sample. In
still another embodiment, the control unit can be configured to
measure the initial fill velocity of the sample directly after the
sample enters a capillary space of the electrochemical cell. In yet
another embodiment, the control unit can be configured to measure
the initial fill velocity after the sample crosses into a region of
a capillary space of the electrochemical where a detection signal
is generated. At least one of the control unit, the electrochemical
cell, and the meter can be configured to measure a temperature of
the sample or infer a temperature of the sample.
[0021] One exemplary embodiment of a method for measuring an
antigen in a blood sample can include providing an immunosensor
having two electrodes and a meter configured to apply a potential
between the two electrodes of the immunosensor. The method can
further include introducing a blood sample including an antigen
into the immunosensor, applying an electric potential between the
two electrodes, determining an initial fill velocity of the blood
sample, and calculating a concentration of the antigen in view of
the initial fill velocity. In an alternative embodiment, the method
can be set-up to only measure a hematocrit level of the blood, or
to measure both a hematocrit level of the blood and a concentration
of the antigen in the blood. The immunosensor can further include a
reaction chamber and a detection chamber formed in a separator
disposed between the two electrodes, a fill chamber at least
partially formed in the separator and one of the two electrodes,
and a vent at least partially formed in the separator and the two
electrodes. The fill chamber can be spaced a distance apart from
the detection chamber and can overlap at least a portion of the
reaction chamber. The vent can be spaced a distance apart from the
reaction chamber and can overlap at least a portion of the
detection chamber. The antigen of the blood sample can be
C-reactive protein. The method can further include measuring a
temperature of the blood sample, or alternatively inferring a
temperature of the blood sample, and then measuring a change in
current over a period of time after reversing the electric
potential. As a result, a concentration of the antigen can be
calculated in view of the change in current over the period of time
and the measured or inferred temperature.
[0022] The method for measuring a blood sample can further include
providing an antibody-enzyme conjugate in a first buffer and
magnetic beads linked to an antigen in a second buffer in the
reaction chamber. Ferricyanide, glucose, and a mediator in a dilute
acid can be provided in the detection chamber. A first seal can be
provided over a first side of the vent that forms a wall of the
fill chamber and a second seal can be provided over a second side
of the vent. At least a portion of the blood sample that is
introduced into the immunosensor moves from the fill chamber to the
reaction chamber when it is introduced into the immunosensor.
[0023] The method can further include opening the vent after a
pre-determined time by piercing at least one of the seals. Piercing
at least one of the seals allows portions of the blood sample
containing the antibody-enzyme conjugate that are not bound to the
magnetic beads to move to the detection chamber. Still further, the
method can include catalyzing oxidation of the glucose in the
detection chamber, which can result in the formation of
ferrocyanide. A current can be electrochemically detected from the
ferrocyanide, and a concentration of the antigen in the blood
sample can be calculated in view of the signal detected.
[0024] In one embodiment, determining an initial fill velocity can
include measuring an initial current after applying the electric
potential, determining a level of hematocrit in the sample, and
reversing the electric potential between the working and counter
electrodes. Accordingly, the concentration of the analyte can be
computer based on the determined level of hematocrit. The method
can further include measuring a change in current over a period of
time following reversing the electric potential. Accordingly, the
concentration of the analyte can be calculated in view of the
change in current over the period of time. In another embodiment,
determining an initial fill velocity can include determining a rate
of change in an optical signal to determine the initial fill
velocity. In still another embodiment, determining an initial fill
velocity can include determining an initial current flow to
determine the initial fill velocity. The initial fill velocity can
be determined directly after the blood sample enters a capillary
space of the immunosensor. Alternatively, the initial fill velocity
can be determined after the blood sample crosses into a region of a
capillary space of the immunosensor where a detection signal is
generated.
BRIEF DESCRIPTION OF DRAWINGS
[0025] This invention will be more fully understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which:
[0026] FIG. 1 illustrates a perspective view of one exemplary
embodiment of an immunosensor and a control unit having an optical
detector for calculating an initial fill velocity in accordance
with the present invention;
[0027] FIG. 2 illustrates an exploded view of another exemplary
embodiment of an immunosensor in accordance with the present
invention, wherein the immunosensor is configured for use with a
control unit having an electrochemical detection system for
calculating an initial fill velocity;
[0028] FIG. 3 illustrates a side elevation schematic drawing (not
to scale) of an exemplary embodiment of an electrochemical cell in
accordance with the present invention;
[0029] FIG. 4 illustrates a plan view, from above, of the
electrochemical cell of FIG. 3;
[0030] FIG. 5 illustrates a schematic drawing (not to scale), in
cross-section, of an exemplary embodiment of a hollow
electrochemical cell in accordance with the present invention;
[0031] FIG. 6 illustrates a plot of a current versus time transient
performed using the device of FIG. 2 in conjunction with one
exemplary example for testing a variety of blood samples provided
herein;
[0032] FIG. 7 illustrates a plot of a hematocrit concentration
level for each blood sample used in association with the example
associated with FIG. 6 versus a current;
[0033] FIG. 8 illustrates a plot of a percent error of the
determined hematocrit concentration levels for each blood sample
associated with FIG. 6 versus the determined hematocrit
concentration levels of each blood sample associated with FIG.
6;
[0034] FIG. 9 illustrates a plot of a calculated C-reactive protein
level of each blood sample associated with FIG. 6 versus a
reference value of plasma C-reactive protein as determined by a
conventional enzyme immunoassay;
[0035] FIG. 10 illustrates a plot of a current versus a temperature
of a detection chamber of the immunosensor in which the blood
samples are disposed performed using the immunosensor of FIG. 2 in
conjunction with another exemplary example for testing a variety of
blood samples provided herein;
[0036] FIG. 11 illustrates a plot of a percent error of the
determined hematocrit concentration levels for each blood sample
associated with FIG. 10 versus the determined hematocrit
concentration levels of each blood sample associated with FIG.
10;
[0037] FIG. 12 illustrates a plot of a determined slope based on a
change in current over time for each blood sample associated with
FIG. 10 versus a temperature of a detection chamber of the
immunosensor in which the blood samples are disposed; and
[0038] FIG. 13 illustrates a plot of a calculated C-reactive
protein level of blood samples associated with FIG. 10 having
approximately a 33.5% hematocrit level and approximately a 47.5%
hematocrit level versus a reference value of plasma C-reactive
protein as determined by a conventional enzyme immunoassay.
DETAILED DESCRIPTION
[0039] Certain exemplary embodiments will now be described to
provide an overall understanding of the principles of the
structure, function, manufacture, and use of the devices and
methods disclosed herein. One or more examples of these embodiments
are illustrated in the accompanying drawings. Those skilled in the
art will understand that the devices and methods specifically
described herein and illustrated in the accompanying drawings are
non-limiting exemplary embodiments and that the scope of the
present invention is defined solely by the claims. The features
illustrated or described in connection with one exemplary
embodiment may be combined with the features of other embodiments.
Such modifications and variations are intended to be included
within the scope of the present invention. Further, while some
embodiments discuss determining a value of hematocrit of a sample
while other embodiments discuss determining a concentration of an
analyte in a sample, one skilled in the art will recognize that the
teachings associated with each type of embodiment are equally
applicable to the other type of embodiment. That is, embodiments
directed to determining hematocrit values can also be used to
determine a concentration of an analyte in a sample, and
embodiments directed to determining a concentration of an analyte
can be used solely to determine a hematocrit value of a sample.
Further, embodiments can both be used to determine a hematocrit
value of a sample and determine a concentration of an analyte in a
sample.
[0040] The methods for determining a value of hematocrit in a
sample and determining a concentration of an analyte in a sample
disclosed herein can be used with any sample analyzing device
and/or system. The devices can have a capillary space. The devices
can include at least one working electrode and one counter
electrode between which an electric potential can be applied. The
sample analyzing device can generally be associated with a
component for applying the electric potential between the
electrodes, such as a meter. The sample analyzing device can also
be associated with one or more components that are capable of
measuring an initial fill velocity of a sample when it is
introduced to the device. Such components can also be capable of
calculating a concentration of an analyte in the sample in view of
the initial fill velocity. Such components are generally referred
to herein as control units. Further, the terms analyte, antigen,
and antibodies are used interchangeably within, and thus, use of
one term is equally applicable to all three terms, unless otherwise
indicated or reasonably known by one skilled in the art.
[0041] In one exemplary embodiment of a method for determining a
hematocrit value of a whole blood sample, a sample of whole blood
is provided to a sample analyzing device having a capillary space.
An initial fill velocity of the sample in at least a portion of the
capillary is measured. A hematocrit value of the sample is then
determined from the initial fill velocity. A concentration of an
analyte or antigen in the sample can be determined in view of the
determined value of hematocrit. Using the initial fill velocity to
calculate the hematocrit value can allow for improved accuracy.
Methods for determining a hematocrit value can also account for the
effects of temperature, as discussed in greater detail below.
Further, by measuring for only a value of hematocrit, without
reference to an associate analyte concentration, determinations can
be achieved almost instantaneously, often in less than a second.
For example, hematocrit levels of a drop of blood can be determined
in less than a second merely by dropping the blood onto a sensor
strip of a sample analyzing device. Once the blood is disposed on
the strip, a digital readout of the hematocrit level can be
provided almost instantaneously. The result is quick and accurate
determinations of hematocrit levels, which are useful for a variety
of medical assessments, for example, making assessments related to
conditions such as anemia.
[0042] In another exemplary embodiment of a method for determining
a concentration of an analyte in a sample, a sample is provided to
a sample analyzing device that has a working electrode and a
counter electrode. An electric potential can be applied between the
working and counter electrodes of the sample analyzing device and
an initial fill velocity of the sample into a capillary space of
the sample analyzing device can be determined. A concentration of
the analyte in the sample can be calculated in view of the
determined initial fill velocity. By calculating the concentration
in view of the initial fill velocity, errors, such as those that
can result from varying hematocrit levels across samples, can be
accounted for, thereby leading to more accurate determinations of
the concentrations of the analytes in the samples. Methods can also
account for the effects of temperature, as discussed in greater
detail below. In an alternative embodiment for detecting a
concentration of an analyte in a sample, errors are corrected for
based on a determined fill time rather than a determined initial
fill velocity. One example of such a device is disclosed in a
co-pending patent application entitled "Systems, Devices, and
Methods for Improving Accuracy of Biosensors Using Fill Time," of
Ronald C. Chatelier and Alastair M. Hodges, filed concurrently with
the present application on Dec. 30, 2009, and issued as U.S. Pat.
No. 8,101,065, which is hereby incorporated by reference in its
entirety. In an alternative embodiment, a concentration of an
antigen in a plasma phase and an estimate of a level of hematocrit
level can be determined.
[0043] An initial fill velocity can be used in a variety of ways to
determine a concentration of an analyte. For example, if the sample
includes whole blood and a temperature of the location where the
sample is being analyzed in the sample analyzing device is known,
the initial fill velocity can be linked to the determined
hematocrit level. A temperature of the sample may be known, for
example, if a chamber of a sample analyzing device is preheated to
a desired temperature. If a temperature is not known, calculations
can still be performed that allow for the temperature to be
measured or inferred during reactions. In such an instance, the
temperature and hematocrit levels can both be accounted for in
order to provide more accurate analyte concentration
determinations. Further, an initial fill velocity can likewise be
used in a variety of ways to determine a hematocrit level of a
blood sample.
[0044] There are a variety of ways to determine the initial fill
velocity associated with the sample entering the sample analyzing
device. Determining the initial fill velocity, in turn, can allow a
viscosity of a liquid to be estimated. Estimating a viscosity of a
liquid can assist in making more accurate concentration
determinations. In one exemplary embodiment, as shown in FIG. 1, an
immunosensor 10 includes a control unit 50 having an optical
detector 52 generally located near an entry port 21 to a fill
chamber 22 of the immunosensor 10. The optical detector 52 can have
any shape or size, and can be located, for example, on top of the
immunosensor 10 or just inside of the entry port 21 of the
immunosensor 10. In the illustrated embodiment, the optical sensor
is coupled to a top plate 14 of the immunosensor 10, adjacent the
entry port 21. The optical sensor 52 can include an optical signal
that changes when a sample passes by the sensor 52. Thus, as a
sample is provided to the immunosensor 10, a rate of change of the
optical signal can be detected, which in turn can be used to
estimate the initial fill velocity. The rate of change can be
measured in at least a portion of a capillary space of the
immunosensor 10. The initial fill velocity can then be used to
calculate a number of different parameters. By way of non-limiting
example, the initial fill velocity can be used to calculate a
concentration of an antigen in a sample or a hematocrit level of a
whole blood sample.
[0045] In another exemplary embodiment, an electrochemical
detection system can be used to measure a magnitude of an initial
current flow. The magnitude can be measured as soon as the sample
enters a capillary space of the sample analyzing device. Capillary
space can be located, for example, prior to an initial entrance
into a fill chamber, between a fill chamber and a reaction chamber,
and/or between a reaction chamber and a detection chamber. In one
exemplary embodiment, the initial current flow is determined
between the fill chamber and the reaction chamber. In another
exemplary embodiment, the initial current flow is measured when the
sample first crosses into a region of capillary space of the sample
analyzing device where a detection signal can be generated, such as
a detection chamber.
[0046] A number of different techniques can be used to measure the
current flow. For example, a desired number of measurements can be
taken over a desired length of time. In one exemplary embodiment, a
measurement is made approximately in the range of about every 1
millisecond to about every 25 milliseconds over a period of
approximately at least about 10 milliseconds to about 300
milliseconds. In another embodiment, a measurement is made
approximately every 10 milliseconds over a period of approximately
at least 50 milliseconds. A single measurement can also be taken,
but typically more accurate results for the initial velocity can be
obtained by making multiple measurements over a short period of
time. One skilled in the art will recognize that there are a
variety of other ways by which the initial current and/or initial
velocity of the sample can be determined, some of which are
disclosed in greater detail below.
[0047] Another exemplary embodiment of a sample analyzing device
for use in conjunction with at least some of the methods disclosed
herein, an immunosensor 110, is illustrated in FIG. 2 and is
described in U.S. patent application Ser. No. 12/570,268 of
Chatelier et al., entitled "Adhesive Compositions for Use in an
Immunosensor" and filed on Sep. 30, 2009, the contents of which is
hereby incorporated by reference in its entirety. A plurality of
chambers can be formed within the immunosensor, including a fill
chamber, by which a sample can be introduced into the immunosensor,
a reaction chamber, by which a sample can be reacted with one or
more desired materials, and a detection chamber, by which a
concentration of a particular component of the sample can be
determined. These chambers can be formed in at least a portion of a
lower electrode, an upper electrode, and a separator of the
immunosensor. The immunosensor can also include a vent hole to
allow air to enter and escape the immunosensor as desired, and
first and second sealing components to selectively seal first and
second sides of the vent hole. The first sealing component can also
form a wall of the fill chamber.
[0048] As illustrated, the immunosensor 110 includes a lower
electrode 112 having two liquid reagents 130, 132 striped onto it.
The lower electrode 112 can be formed using any number of
techniques used to form electrodes, but in one embodiment a
polyethylene terephthalate (PET) sheet that is filled with barium
sulphate is sputter-coated with a suitable conductor, such as, for
example, gold. Other non-limiting example of forming an electrode
are disclosed in U.S. Pat. No. 6,521,110 of Hodges et al., entitled
"Electrochemical Cell" and filed on Nov. 10, 2000, the contents of
which is hereby incorporated by reference in its entirety.
[0049] Likewise, the liquid reagents 130, 132 may have a number of
different compositions. In one embodiment, the first liquid reagent
130 includes an antibody conjugated to an enzyme, such as, for
example, GDH-PQQ, in a buffer that contains sucrose, as well as a
poloxamer, such as, for example, Pluronics.RTM. block copolymers,
an anticoagulant, such as citraconate, and calcium ions. In one
embodiment, the second liquid reagent 132 includes a mixture of
ferricyanide, glucose, and a second mediator, such as phenazine
ethosulfate, in an acidic buffer, such as a dilute citraconic acid
solution. The first and second liquid reagents 130, 132 can be
dried onto the lower electrode 112. A number of techniques can be
used to dry the reagents 130, 132, but in one embodiment, following
the striping of the reagents 130, 132 on the lower electrode 112,
one or more infrared dryers can be applied to the reagents 130,
132. One or more air dryers can also be used, for example,
subsequent to the infrared dryers. References to a first reagent
and a first liquid reagent and a second reagent and a second liquid
reagent herein are used interchangeably and are not necessarily an
indication that the reagents are in their liquid or dried form at a
given time for a particular embodiment. Further, some of the
components associated with the first and second liquid reagents can
be used interchangeably and/or in both the first and second liquid
reagents as desired. By way of non-limiting example, an
anticoagulant can be associated with either or both of the first
liquid reagent 130 and the second liquid reagent 132.
[0050] A line can be formed in the sputter-coated gold between the
reagents 130, 132 such that an edge of one of the reagents 130, 132
is very close to, or touches, the line. In the illustrated
embodiment, the line is formed such that an edge of the reagent 132
touches the line at vent 124. The line can be applied using laser
ablation or with a sharp metal edge. In one exemplary embodiment,
the line can be applied before the reagents 130, 132 are striped on
the electrode. The line can be designed to electrically insulate
the section of the lower electrode 112 under the detection chamber
from the section that will be under the reaction chamber. This can
provide a better definition of an area of the working electrode
during the electrochemical assay.
[0051] The immunosensor 110 can also include an upper electrode 114
having one or more magnetic beads 134 containing surface-bound
antigens thereon. The antigens can be configured to react with the
antibody disposed on the lower electrode 112 and the sample within
a reaction chamber 118, as described in further detail below. One
skilled in the art will recognize that the components disposed on
the lower electrode 112 and on the upper electrode 114 can be
interchangeable. Thus, the lower electrode 112 can include one or
more magnetic beads 134 and the upper electrode 114 can include two
liquid reagents 130, 132 striped onto it. Further, although in the
illustrated embodiment the length of the electrode 112 forms the
length of the entire body of the immunosensor 110, in other
embodiments the electrode can be only a portion of a layer of an
immunosensor that serves as the lower or upper electrode or
multiple electrodes can be disposed on a single layer of an
immunosensor. Further, because potential applied to the
immunosensor can be flipped and/or alternated, each of the lower
and upper electrodes can serve as the working electrode and the
counter or counter/reference electrode at different stages. For
ease of description purposes, in the present application the lower
electrode is considered the working electrode and the upper
electrode the counter or counter/reference electrode.
[0052] A separator 116 disposed between the lower and upper
electrodes 112, 114 can have a variety of shapes and sizes, but it
generally is configured to desirably engage the lower and upper
electrodes 112, 114 to form the immunosensor 110. In one exemplary
embodiment, the separator 116 includes adhesive properties on both
sides. The separator 116 can further include a release liner on
each side of the two sides of the separator 116. The separator 116
can be cut in a manner that forms at least two cavities. A first
cavity can be formed to serve as a reaction chamber 118 and a
second cavity can be formed to serve as a detection chamber 120. In
one embodiment, the separator 116 can be kiss-cut such that the
reaction chamber 118 is aligned with the electrodes 112, 114 to
allow an antigen-antibody reaction therein while the detection
chamber 120 is aligned with the electrodes 112, 114 to allow for
the electrochemical determination of ferrocyanide therein.
[0053] In one embodiment, the separator 116 can be placed on the
lower electrode 112 in a manner that allows the magnetic beads 134
of the upper electrode 114 and the first reagent 130 of the lower
electrode 112 to be at least partially disposed in the reaction
chamber 118 and the ferricyanide-glucose combination of the second
reagent 132 of the lower electrode 112 to be at least partially
disposed in the detection chamber 120. It can be advantageous to
include an anticoagulant in each of the first and second liquid
reagents 130, 132 so that an anticoagulant is associated with each
of the reaction and detection chambers 118, 120. In some
embodiments the combination of one of the upper and lower
electrodes 112, 114 and the separator 116 can be laminated together
to form a bi-laminate, while in other embodiments the combination
of each of the lower electrode 112, the upper electrode 114, and
the separator 116 can be laminated together to form a tri-laminate.
Alternatively, additional layers may also be added.
[0054] A fill chamber 122 can be formed by punching a hole into one
of the lower and upper electrodes 112, 114 and the separator 116.
In the illustrated embodiment, the fill chamber is formed by
punching a hole in the lower electrode 112 and the separator 116
such that the hole in the lower electrode 112 overlaps the reaction
chamber 118. As shown, the fill chamber 122 can be a distance apart
from the detection chamber 120. Such a configuration allows a
sample to enter the immunosensor 110 through the fill chamber 122
and flow into the reaction chamber 118 to be reacted, for example
with the first liquid reagent 130 that includes the antibody
conjugated to an enzyme in a buffer on the first electrode 112 and
the magnetic beads 134 striped on the upper electrode 114, without
entering the detection chamber 120. Entry of a sample into the fill
chamber 122 can occur by way of capillary action, and as such, at
least one of the fill chamber 122, the reaction chamber 118, and a
location therebetween can be considered a capillary space. Once the
sample has been reacted, it can then flow into the detection
chamber 120 for interaction with the second liquid reagent 132, for
example, the mixture of ferricyanide, glucose, and the second
mediator in an acidic buffer.
[0055] A vent 124 can be formed by punching a hole through each of
the two electrodes 112, 114 and the separator 116 such that the
vent 124 extends through the entirety of the immunosensor 110. The
hole can be formed in a suitable manner such as, for example,
drilled or punched in a number of different locations, but in one
exemplary embodiment it can overlap a region of the detection
chamber 120 that is spaced apart from the reaction chamber 118.
[0056] The vent 124 can be sealed in a number of different manners.
In the illustrated embodiment, a first sealing component 140 is
located on the lower electrode 112 to seal a first side of the vent
124 and a second sealing component 142 is located on the upper
electrode 114 to seal a second side of the vent 124. The sealing
components can be made of and/or include any number of materials.
By way of non-limiting example, either or both of the sealing
components can be hydrophilic adhesive tape or Scotch.RTM. tape.
Adhesive sides of the sealing components can face the immunosensor
110. As shown, not only can the first sealing component 140 form a
seal for the vent 124, but it can also form a wall for the fill
chamber 122 so that the sample can be contained therein. Properties
incorporated onto the adhesive side of the first sealing component
140 can be associated with the fill chamber 122. For example, if
the first sealing component 140 includes properties making it
hydrophilic and/or water soluble, the fill chamber can remain
well-wet when a sample is disposed therein. Further, the sealing
components 140, 142 can be selectively associated and disassociated
with the immunosensor 110 to provide venting and/or sealing for the
immunosensor 110 and the components disposed therein as
desired.
[0057] Adhesives can generally be used in the construction of the
immunosensor. Non-limiting examples of ways in which adhesives can
be incorporated into immunosensors and other sample analyzing
devices of the present disclosure can be found in U.S. patent
application Ser. No. 12/570,268 of Chatelier et al., entitled
"Adhesive Compositions for Use in an Immunosensor" and filed on
Sep. 30, 2009, the contents of which was already incorporated by
reference in its entirety.
[0058] While the present disclosure discusses a variety of
different embodiments related to immunosensors, other embodiments
of immunosensors can also be used with the methods of the present
disclosure. Non-limiting examples of such embodiments include those
described in U.S. Patent Application Publication No. 2003/0180814
of Hodges et al., entitled "Direct Immunosensor Assay" and filed on
Mar. 21, 2002, U.S. Patent Application Publication No. 2004/0203137
of Hodges et al., entitled "Immunosensor" and filed on Apr. 22,
2004, U.S. Patent Application Publication No. 2006/0134713 of
Rylatt et al., entitled "Biosensor Apparatus and Methods of Use"
and filed on Nov. 21, 2005, and U.S. patent application Ser. No.
12/563,091, which claims priority to each of U.S. Patent
Application Publication Nos. 2003/0180814 and 2004/0203137, each of
which is hereby incorporated by reference in its entirety.
[0059] In one embodiment, the immunosensor 110 can be configured to
be placed into a meter that is configured to apply a potential to
the electrodes 112, 114 and measure a current that results from the
application of the potential. In one embodiment, the immunosensor
includes one or more tabs 117 for engaging a meter. Other features
can also be used to engage the immunosensor 110 with a meter. The
meter can include a number of different features. For example, the
meter can include a magnet that is configured to maintain certain
components of the immunosensor 110 in one chamber while other
components flow to the other. In one exemplary embodiment, the
magnet of the meter is located such that, upon placing the
immunosensor 110 in the meter, the magnet is disposed below the
reaction chamber 118. This can allow the magnet to assist in
holding back any magnetic beads 134, and more particularly any
antibody-enzyme conjugate that is bound to the beads 134, from
flowing into the detection chamber 120.
[0060] An alternate feature of the meter includes a heating
element. A heating element can help speed up the reaction rate and
help the sample flow through the immunosensor 110 in a desired
manner by reducing the viscosity. A heating element can also allow
one or more chambers and/or a sample disposed therein to be heated
to a predetermined temperature. Heating to a predetermined
temperature can help provide accuracy, for example, by diminishing
or removing the effects of temperature change as reactions
occur.
[0061] Further, a piercing instrument can also be associated with
the meter. The piercing instrument can be configured to pierce at
least one of the first and second sealing components at a desired
time so that air can flow out of the vent hole and liquid can flow
from the reaction chamber into the detection chamber.
[0062] The immunosensor 110 can also be configured to be associated
with a control unit. The control unit can be configured to perform
a variety of functions. In one exemplary embodiment, the control
unit is capable of measuring an initial fill velocity of a sample
when it is introduced to the device. In another embodiment, the
control unit is configured to determine a hematocrit value of a
blood sample. In yet another embodiment, the control unit is
configured to calculate a concentration of an analyte in the sample
in view of the initial fill velocity. In fact, the control unit can
include a number of different features, depending, at least in
part, on the functionality desired and the method by which the
system is designed to measure the initial fill velocity.
[0063] By way of non-limiting example, if the system is designed to
measure an initial fill velocity optically, the control unit can
include an optical signal detector. The optical signal detector can
measure an initial fill velocity based on a rate of change in an
optical signal sensed by the detector. Alternatively, if the system
is designed to measure an initial fill velocity based on current
flow, the control unit can include a current flow detector. The
current flow detector can measure an initial fill velocity based on
a change in current that occurs as a result of the sample entering
the immunosensor. The timing of this change can occur in a number
of different manners, but in one exemplary embodiment, the current
is measured after the sample crosses into a region of a capillary
space of the immunosensor where a detection signal is generated,
for example, when the sample crosses from the reaction chamber into
the detection chamber. In another embodiment, the current is
measured directly after the sample enters a capillary space of the
immunosensor, for example, when the sample enters the reaction
chamber.
[0064] The control unit can also measure other aspects of the
system. By way of non-limiting example, the control unit can be
configured to measure a temperature of one or more chambers of the
immunosensor. It can also be configured to measure a temperature of
the sample, for instance directly or by measuring an ambient
temperature and using it to infer the temperature of the sample, a
color of the sample, or a variety of other characteristics and/or
properties of the sample and/or the system. By way of further
non-limiting example, the control unit can be configured to
communicate the results of the initial fill velocity determination,
the results of the hematocrit value determination, and/or the
results of the analyte concentration determination, to outside
equipment. This can be accomplished in any number of ways. In one
embodiment, the control unit can be hardwired to a microprocessor
and/or a display device. In another embodiment, the control unit
can be configured to wirelessly transmit data from the control unit
to a microprocessor and/or a display device.
[0065] Other components of the system can also be configured to
make such measurements. For example, the immunosensor or the meter
can be configured to measure a temperature of one or more chambers
of the immunosensor, measure or infer the temperature of a sample,
or measure, determine, or infer a variety of other characteristics
and/or properties of the sample and/or the system. Still further,
one skilled in the art will recognize that these features of a
control unit can be interchanged and selectively combined in a
single control unit. For example, a control unit can both determine
an initial fill velocity and measure a temperature of a chamber. In
other embodiments, multiple control units can be used together to
perform various functions, based at least in part on the
configurations of the various control units and the desired
functions to be performed.
[0066] Other types of sample analyzing devices can be used in
conjunction with at least some of the systems and methods disclosed
herein. These devices can include, by way of non-limiting example,
electrochemical cells, electrochemical sensors, glucose sensors,
glucose meters, hemoglobin sensors, antioxidant sensors, and
biosensors. In one embodiment, the sample analyzing device includes
a glucose sensor. The glucose sensor can include an electrochemical
cell, such as the cell illustrated in FIGS. 3 and 4. The cell can
include a thin strip membrane 201 having upper and lower surfaces
202, 203, and can also include a cell zone 204 defined between a
working electrode 206 disposed on the lower surface 203 and a
counter/reference electrode 205 disposed on the upper surface 202.
The membrane thickness can be selected to achieve a desired result,
such as having the reaction products from a counter electrode
arrive at a working electrode. For instance, the membrane thickness
can be selected so that the electrodes are separated by a distance
t, which can be sufficiently close such that the products of
electrochemical reaction at the counter electrode can migrate to
the working electrode during the time of the test and a steady
state diffusion profile can be substantially achieved. Typically t
can be less than approximately 500 micrometers, alternatively in
the range of about 10 micrometers to about 400 micrometers, and
more particularly in the range of about 80 micrometers to about 200
micrometers. In one embodiment, a spacing between the electrodes
can be selected such that the reaction products from a counter
electrode arrive at a working electrode.
[0067] The electrodes can also have a variety of configurations.
For instance, the electrodes can be planar. Further, while in the
illustrated embodiment the electrodes 205, 206 are facing each
other and are substantially opposed, in other embodiments the
electrodes can just be facing each other, they can be substantially
opposed to each other, or they can have a side-by-side
configuration in which the electrodes are positioned approximately
in the same plane. Examples of different electrode configurations
can be found at least in U.S. Pat. No. 7,431,820 of Hodges,
entitled "Electrochemical Cell," and filed on Oct. 14, 2003, the
contents of which is hereby incorporated by reference in its
entirety.
[0068] A sample deposition or "target" area 207 can be defined on
the upper surface 202 of the membrane 201 and can be spaced at a
distance greater than the membrane thickness from the cell zone
204. The membrane 201 can have a diffusion zone 208 that can extend
between the target area 207 and the cell zone 204. A suitable
reagent can include a redox mediator M, an enzyme E, and a pH
buffer B, each of which can be contained within the cell zone 204
of the membrane and/or between the cell zone 204 and the target
area 207. The reagent can also include stabilizers and the
like.
[0069] In use of the sensor, a drop of blood can be placed on the
target zone 207 and the blood components can wick towards the cell
zone 204. The initial velocity at which the blood covers the target
zone 207 can depend at least on the hematocrit.
[0070] Each of electrodes 205, 206 can have a predefined area. In
the embodiments of FIGS. 3 and 4 the cell zone 204 can defined by
edges 209, 210, 211 of the membrane, which can correspond with
edges of the electrodes 205, 206 and by leading (with respect to
the target area 207) edges 212, 213 of the electrodes. In the
present example the electrodes can be about 600 angstrom thick and
can be from about 1 mm to about 5 mm wide, although a variety of
other dimensions and parameters can be used without departing from
the scope of the present invention.
[0071] Alternatively, both sides of the membrane can be covered
with the exception of the target area 207 by laminating layers
which can serve to prevent evaporation of water from the sample and
to provide mechanical robustness to the apparatus. Evaporation of
water is believed to be undesirable as it concentrates the sample,
allows the electrodes to dry out, and allows the solution to cool,
affecting the diffusion coefficient and slowing the enzyme
kinetics, although diffusion coefficient can be estimated as
above.
[0072] In an alternative embodiment, illustrated in FIG. 5, a
hollow electrochemical cell for use with the systems and methods
disclosed herein is provided. The electrodes 305, 306 can be
supported by spaced apart polymer walls 330 to define a hollow
cell. An opening 331 can be provided on one side of the cell
whereby a sample can be admitted into the cavity 332. In this
embodiment, a membrane is not used, although in some embodiments a
membrane can be included. The electrodes can have a variety of
configurations, at least as discussed above. By way of non-limiting
example, the electrodes can be spaced apart by less than about 500
micrometers, preferably in the range of about 10 micrometers or
about 20 micrometers to about 400 micrometers, and more preferably
in a range of about 100 micrometers to about 200 micrometers. The
effective cell volume can be about 1.5 microliters or less.
[0073] The electrochemical cells of FIGS. 3-5 can be used in
conjunction with the meters, control units, and other components
and steps of the devices, systems, and methods disclosed herein.
Further disclosures related to the electrochemical cells of FIGS.
3-5 are found in U.S. Pat. No. 6,284,125 of Hodges et al., entitled
"Electrochemical cell" and filed on Apr. 17, 1998, the contents of
which is hereby incorporated by reference in its entirety. For
example, electrochemical cells used in conjunction with the present
disclosures can have two electrode pairs. The electrode pairs can
include any combination of working, counter, counter/reference, and
separate reference electrodes.
Example 1
[0074] The use of an electrochemical system to measure an initial
fill velocity based on measuring current flow is demonstrated by
the following example. In the following example, the system
included a sample analyzing device, in particular the immunosensor
110 of FIG. 2, a meter configured to apply a potential, and a
control unit configured to determine the initial fill velocity. In
particular, a potential was applied to the electrodes of the
immunosensor 110, a level of hematocrit was determined, and then
the potential was reversed. The concentration of the analyte was
subsequently determined in view of the determined level of
hematocrit. The level of hematocrit was determined in view of a
calculated initial fill velocity.
[0075] A plurality of samples were provided for analysis to test
the performance of the systems, devices, and methods disclosed
herein. The samples were blood samples that contained C-reactive
proteins, and thus the concentration of the analyte being
determined was the concentration of C-reactive proteins. The
samples contained four different levels of hematocrit, which were
known so comparisons of the test results could be compared to the
actual results to determine the accuracy of the systems, devices,
and methods. The four levels of hematocrit were approximately 33%,
approximately 41.5%, approximately 47.5%, and approximately 55%.
Testing four levels of hematocrit allowed the accuracy of the
disclosed systems, devices, and methods to be confirmed over a
broad spectrum of concentration levels.
[0076] In this first example, an immunosensor was preheated to
approximately 37.degree. C. before a sample was introduced. The
meter associated with the immunosensor was configured to perform
the preheating, although other alternatives could have been used.
Samples were then introduced into the immunosensor. While the
introduction of samples into the immunosensor could have been
accomplished in a variety of manners, in the example each sample
was admitted individually by way of capillary action into the fill
chamber.
[0077] After approximately two minutes had elapsed, the vent of the
immunosensor was accessed by piercing the first sealing component.
A piercing instrument of the meter was used to perform the piercing
action, which in turn allowed the blood to flow from the reaction
chamber of the immunosensor into the detection chamber of the
immunosensor. As soon as the blood started to enter the detection
chamber, a potential of about 300 mV was applied to the electrodes
by way of the meter for approximately four seconds. Alternatively,
the potential could have been applied prior to or while the blood
was arriving in the detection chamber. Subsequently, the potential
was interrupted and reversed for approximately 10 seconds. A plot
of the current versus time transient resulting from this example is
illustrated in FIG. 6. The initial current for each sample, which
in the present example was measured about every 10 milliseconds and
then averaged over about the first 50 milliseconds, is related to
the hematocrit level of the particular sample. A level of
hematocrit is determined from the initial current during the first
application of electric potential, while a level of C-reactive
protein is calculated following the reversed potential, based on
the slope of the current versus time plot and the determined level
of hematocrit.
[0078] As discussed above, in some embodiments it may be desirable
to only measure a level of hematocrit. Thus, the first calculation
based on the initial current may be the only step that is needed to
make that calculation. While in the present example this
determination is made as a result of a four second potential
application, the actual determination of the hematocrit level can
be determined as quickly as the initial current can be calculated.
Thus, by way of non-limiting example, if the initial current is
calculated based on an average over about the first 50
milliseconds, the level of hematocrit can be determined following
about the first 50 milliseconds. Thus, measurements of a hematocrit
level of a blood sample can be performed in less than one
second.
[0079] The level of hematocrit for each sample that was determined
is illustrated by FIG. 7. FIG. 7 illustrates a plot of the
concentration level of the hematocrit for each sample versus the
determined initial current. The plot clearly shows that samples
containing four different levels of hematocrit were tested, which
correlates with the known concentration levels. Further, as
illustrated, higher levels of hematocrit generally led to lower
absolute values of the measured initial currents. For example,
samples having a concentration of hematocrit that was approximately
33% had initial current absolute values that were approximately in
the range of about 38 microamperes to about 33 microamperes, while
samples having a concentration of hematocrit that was approximately
47.5% had initial current absolute values that were approximately
in the range of about 31 microamperes to about 26 microamperes. A
best fit line of all of the results was determined, which is also
illustrated in FIG. 7. The equation that correlates with the best
fit line is:
H=97.6-1.7658|i.sub.i| (Eq. 1)
where H is the level of hematocrit and |i.sub.i| is the initial
current. The error between the equation that illustrates the
results of the hematocrit level versus initial current and the
actual results is illustrated in FIG. 8. More particularly, FIG. 8
plots the percent error that existed in each test sample versus the
actual measured hematocrit level. Every actual result but two was
within about .+-.5% of the calculated range, with a substantial
amount in the range of about .+-.2.5%.
[0080] Once the hematocrit level was determined, that result, along
with the slope of the current versus time transient of FIG. 6
approximately between about 9 seconds and about 14 seconds, was
used to calculate the value of C-reactive protein in the sample.
The level of C-reactive protein was determined by the equation:
C.sub.O=-3.5+0.866exp(y) (Eq. 2)
where C.sub.O is the concentration of C-reactive protein and y is
based on the aforementioned slope and the level of hematocrit. More
particularly, y removed the effect of hematocrit on the slope and
was calculated by the following equation:
y = m ( 1 - 0.01 H ) 0.83 ( Eq . 3 ) ##EQU00003##
where m is the slope of the current versus time transient
approximately between about 9 seconds and about 14 seconds and H is
the determined hematocrit level. FIG. 9 illustrates a plot of the
calculated C-reactive protein level of each of the samples versus
the reference value of plasma C-reactive protein as determined by a
conventional enzyme immunoassay. The best fit line in FIG. 9
illustrates an accurate correlation between the determined level of
C-reactive protein and the equivalent reference value.
Example 2
[0081] The use of an electrochemical system to measure an initial
fill velocity based on measuring current flow was further
demonstrated by another example. The sample analyzing device that
was used in this example was also the immunosensor 110 of FIG. 2, a
meter configured to apply a potential, and a control unit
configured to determine the initial fill velocity. In particular, a
potential was applied to the electrodes of the immunosensor 110, a
level of hematocrit was determined, and then the potential was
reversed. The concentration of the analyte was subsequently
calculated in view of the determined level of hematocrit. Similar
to the previous example, a number of samples having varying
hematocrit levels were used with the system in order to demonstrate
the capabilities of the system. The known levels of hematocrit
concentration were approximately 33.5%, approximately 41%,
approximately 47.5%, and approximately 56.5%.
[0082] A sample was introduced into an unheated immunosensor by way
of capillary action. The sample entered the fill chamber and moved
to the reaction chamber, where it remained for approximately five
minutes. The vent of the immunosensor was subsequently opened by
piercing the first sealing component, thereby allowing the blood of
the sample disposed in the immunosensor to flow from the reaction
chamber of the immunosensor into the detection chamber of the
immunosensor. Allowing the sample to wait longer before piercing at
least one of the sealing components provided adequate time for the
antigen and the antibody-enzyme conjugate of the immunosensor to
diffuse and react, particularly in view of the unheated reaction
chamber. Preheating the immunosensor can speed this time up, as
demonstrated by Example 1 above. In the present example, however,
no heating component was included, which provided the benefits of
eliminating complications and costs associated with incorporating a
heating element with the system. In such instances where a
temperature of a chamber is not known or constant, however, the
calculations performed to determine levels of hematocrit and/or
levels of C-reactive protein should account for the effect of
different ambient temperatures in order to provide more accurate
results. Such accounting was provided for in this second example.
In one embodiment, the temperature of the sample can be
inferred.
[0083] Similar to the earlier example, as the blood started to
enter the detection chamber, a potential of approximately 300 mV
was applied to the electrodes by way of the meter for approximately
4 seconds. Subsequently, the potential was interrupted and reversed
for approximately 10 seconds. A plot of the resulting current
versus time transient was created in a manner similar to the plot
illustrated in FIG. 6. From the resulting plot, a level of
hematocrit was determined from the initial current during the first
application of electric potential. Subsequently, a level of
C-reactive protein was calculated following the reversed potential.
The calculated level of C-reactive protein was based on the slope
of the current versus time plot and the determined level of
hematocrit. Accounting for the temperature in this example provided
further accuracy, as shown below.
[0084] The initial current that was determined for each sample is
illustrated by FIG. 10. FIG. 10 illustrates a plot of the
determined initial current versus the temperature of the detection
chamber of the immunosensor in which the sample was disposed. The
initial currents for the four types of samples (i.e., the four
different levels of hematocrit) were measured over a range of
approximately 20.degree. C. to approximately 37.degree. C.
Generally, higher levels of hematocrit led to lower absolute values
of the initial current. As temperatures in the chamber increased,
the absolute values of the initial current also generally
increased. As shown, the initial current varied linearly with
temperature when the hematocrit was fixed. In view of the
temperature of the chamber and the initial current, the level of
hematocrit was determined by the following equation:
H=77.1-2.1|i.sub.i|+0.75T (Eq. 4)
where H is the level of hematocrit, |i.sub.i| is initial current,
and T is the temperature of the detection chamber. Similar to the
earlier example, the errors in the estimated levels of hematocrit
were approximately within .+-.5%, as shown in FIG. 11. FIG. 11
plots the percent error that existed in each test sample versus a
reference hematocrit level of that sample. Also similar to the
earlier example, in some embodiments only a hematocrit value
determination is made, thereby allowing for quick assessments of
various medical conditions that can be evaluated based on
hematocrit value determinations.
[0085] Once the hematocrit level was determined, that result, along
with the slope of the current versus time transient approximately
from about 9 seconds to about 14 seconds and the temperature of the
detection chamber, were used to calculate the value of C-reactive
protein in the sample. The level of C-reactive protein was
determined by the equation:
C.sub.O=5.7+1.78exp(y') (Eq. 5)
where C.sub.O is the concentration of C-reactive protein and y' is
based on the temperature of the detection chamber and a variable y,
which in turn is based on the aforementioned slope and the level of
hematocrit. More particularly, y' removed the effect of temperature
on slope and was calculated by the following equation:
y ' = y 1 + 0.068 ( T - 25 ) ( Eq . 6 ) ##EQU00004##
where T is the temperature of the detection chamber of the
immunosensor and y is a term that removes the effect of hematocrit
on the slope. The equation for y' assumes that the slope changes by
a certain percentage, typically approximately in the range of about
four to about seven percent, for approximately every one degree
.degree. C. change in temperature. Further, the term T-25 corrects
all values of y' to a standard temperature of 25.degree. C. If a
different temperature should be corrected for, this term can be
adjusted accordingly. In fact, one skilled in the art will
recognize many ways to manipulate this equation, and the other
equations disclosed throughout this disclosure, for other samples,
temperatures, etc.
[0086] The variable y was calculated by the following equation:
y = m ( 1 - 0.01 H ) 1.55 ( Eq . 7 ) ##EQU00005##
where m is the slope of the current versus time transient
approximately between about 9 seconds and about 14 seconds and H is
the determined hematocrit level. The term (1-0.01H) represents a
fraction of the volume that is plasma that is then raised to an
arbitrary power. The power can be obtained as a calibration
coefficient.
[0087] The slope of the transient approximately between about 9
seconds and about 14 seconds was a function of C-reactive protein,
a hematocrit level, and the temperature. When the concentration of
C-reactive protein was fixed at approximately 0.15 mg/L, there was
still a considerable variation of the slope with respect to
hematocrit and temperature, as shown in FIG. 12. FIG. 12
illustrates a plot of the determined slope versus the temperature
of the detection chamber of the immunosensor in which the sample
was disposed. Initial currents for each of the four hematocrit
level samples were measured over a range of approximately
20.degree. C. to approximately 37.degree. C. Generally, the greater
the level of hematocrit in a sample, the lower the value of the
slope. As temperatures in the chamber increased, the values of the
slope generally increased.
[0088] FIG. 13 illustrates a plot of the calculated C-reactive
protein level of each of the samples having a hematocrit level of
approximately either about 33.5% or about 47.5%, versus the
reference value of plasma C-reactive protein as determined by a
conventional enzyme immunoassay. The best fit line in FIG. 13
illustrates an accurate correlation between the determined level of
C-reactive protein and the equivalent reference value.
[0089] One skilled in the art will appreciate that these two
examples are merely two of many examples of how the teachings
contained herein can be performed and used. Further, although the
methods, systems, and devices disclosed herein are primarily used
in conjunction with determining a concentration of an analyte of a
blood sample, and are primarily focused on accounting for errors
that can result from varying levels of hematocrit in blood samples,
one skilled in the art will recognize that the disclosures
contained herein can also be used for a variety of other samples
containing analytes and can test for a variety of antigens and/or
antibodies contained within a sample.
[0090] One skilled in the art will also recognize that to the
extent various methods, systems, and devices rely on a particular
equation, the equations provided are generally based on the
examples to which the equations were applied. One skilled in the
art, in view of the present disclosure, will be able to make
adjustments to the disclosed equations for other situations without
departing from the scope of the invention.
[0091] Still further, the methods discussed herein, such as those
related to determining a concentration and using the systems and
devices, are also not limited by the particular steps or order of
the steps, except where indicated. One skilled in the art will
recognize various orders in which the methods can be performed, and
further, will recognize that steps can be modified or added without
departing from the scope of the invention.
[0092] Non-limiting examples of some of the other types of devices
with which the methods disclosed herein can be used are discussed
in greater detail in U.S. Pat. No. 5,942,102 of Hodges et al.,
entitled "Electrochemical Method" and filed on May 7, 1997, U.S.
Pat. No. 6,174,420 of Hodges et al., entitled "Electrochemical
Cell" and filed on May 18, 1999, U.S. Pat. No. 6,379,513 of
Chambers et al., entitled "Sensor Connection Means" and filed on
Sep. 20, 1999, U.S. Pat. No. 6,475,360 of Hodges et al., entitled
"Heated Electrochemical Cell" and filed on Sep. 11, 2000, U.S. Pat.
No. 6,632,349 of Hodges et al, entitled "Hemoglobin Sensor" and
filed on Jul. 14, 2000, U.S. Pat. No. 6,638,415 of Hodges et al.,
entitled "Antioxidant Sensor" and filed on Jul. 14, 2000, U.S. Pat.
No. 6,946,067 of Hodges et al., entitled "Method of Forming an
Electrical Connection Between an Electrochemical Cell and a Meter"
and filed on Dec. 9, 2002, U.S. Pat. No. 7,043,821 of Hodges,
entitled "Method of Preventing Short Sampling of a Capillary or
Wicking Fill Device" and filed on Apr. 3, 2003, and U.S. Pat. No.
7,431,820 of Hodges et al., entitled "Electrochemical Cell" and
filed on Oct. 1, 2002, each of which is hereby incorporated by
reference in its entirety.
[0093] Further, to the extent the disclosures herein are discussed
for use with a device having a particular configuration, any number
of configurations can be used. For example, some configurations
that can be used with the present disclosures include sensors
having two electrodes facing each other, sensors having two
electrodes on the same plane, and sensors having three electrodes,
two of which are opposed and two of which are on the same plane.
These different configurations can occur in any number of devices,
including immunosensors and the other aforementioned devices.
[0094] Various aspects of the devices, systems, and methods can be
adapted and changed as desired for various determinations without
departing from the scope of the present invention. Further, one
skilled in the art will appreciate further features and advantages
of the invention based on the above-described embodiments.
Accordingly, the invention is not to be limited by what has been
particularly shown and described, except as indicated by the
appended claims. All publications and references cited herein are
expressly incorporated herein by reference in their entirety.
* * * * *