IE83676B1 - Method for measuring the concentration of an analyte in whole blood - Google Patents
Method for measuring the concentration of an analyte in whole bloodInfo
- Publication number
- IE83676B1 IE83676B1 IE2002/0900A IE20020900A IE83676B1 IE 83676 B1 IE83676 B1 IE 83676B1 IE 2002/0900 A IE2002/0900 A IE 2002/0900A IE 20020900 A IE20020900 A IE 20020900A IE 83676 B1 IE83676 B1 IE 83676B1
- Authority
- IE
- Ireland
- Prior art keywords
- sample
- matrix
- light
- reading
- wavelength
- Prior art date
Links
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Description
PATENTS ACT , 1992
METHOD FOR MEASURING THE CONCENTRATION OF AN ANALYTE IN
WHOLE BLOOD
LIFESCAN, INC.
METHOD FOR MEASURING THE CONCENTRATION OF AN ANALYTE IN
WHOLE BLOOD
The present invention relates to a method for the
colorimetric determination of chemical and biochemical
components (analytes) in whole blood. In one preferred
embodiment it concerns a method for colorimetrically
measuring the concentration of glucose in whole blood.
The quantification of chemical and biochemical
components in whole blood is of ever—increasing
importance. Important applications exist in medical
diagnosis and treatment and in the quantification of
exposure to therapeutic drugs, intoxicants, hazardous
chemicals and the like. the
In some instances,
concentration of materials being determined are either
so minuscule — in the range of a microgram or less per
deciliter — or so difficult to precisely determine that
the apparatus employed is complicated and useful only
to skilled laboratory personnel. In this case the
results are generally not available for some hours or
days after sampling. In other instances, there is often
an emphasis on the ability of lay operators to perform
the test routinely, quickly and reproducibly outside a
laboratory setting with rapid or immediate information
display.
One common medical test is the measurement of blood
glucose levels by diabetics. Current teaching counsels
diabetic patients to measure their blood glucose
concentration from two to seven times a day depending
on the nature and severity of their individual cases.
Based on the observed pattern in the measured glucose
concentrations the patient and physician together make
adjustments in diet, exercise and insulin intake to
better manage the disease. Clearly, this information
should be available to the patient immediately.
Currently a method widely used in the United States
employs a test article of the type described in U.S.
Patent 3,298,789 issued January 17, 1967 to Mast. In
this method a sample of fresh, whole blood
-40 ul)
(typically
is placed on an ethylcellulose—coated reagent
pad containing an enzyme system having glucose oxidase
and peroxidase activity. The enzyme system reacts with
glucose and releases hydrogen peroxide. The pad also
contains an indicator which reacts with the hydrogen
peroxide in the presence of peroxidase to give a color
proportional in intensity to the sample's glucose
concentration.
Another popular blood glucose test method employs
similar chemistry but in place of the ethylcellulose-
coated pad employs a water—resistant film through which
the enzymes and indicator are dispersed. This type of
system is disclosed in United States Patent 3,630,957
issued December 28, 1971 to Rey et al.
In both cases the sample is allowed to remain in
contact with the reagent pad for a specified time
(typically one minute). Then in the first case the
blood sample is washed off with a stream of water while
in the second case it is wiped off the film. The
reagent pad or film Ls then blotted dry and evaluated.
The evaluation is made either by comparing color
generated with a color chart or by placing the pad or
film in a diffuse reflectance instrument to read a
color intensity value.
While the above methods have been used in glucose
monitoring for years, they do have certain limitations.
The sample size required is rather large for a finger
stick test and is difficult to achieve for some people
whose capillary blood does not express readily.
In addition, these methods share a limitation with
other simple lay—operator colorimetric determinations
in that their result is based on an absolute color
reading which is in turn related to the absolute extent
of reaction between the sample and the test reagents.
The fact that the sample must be washed or wiped off
the reagent pad after the timed reaction interval
requires that the user be ready at the end of the timed
interval and wipe or apply a wash stream at the
required time. The fact that the reaction is stopped by
removing the sample leads to some uncertainty in the
result, especially in the hands of the home user.
Overwashing can give low results and underwashing can
give high results.
Another problem that often exists in simple lay-
operator colorimetric determinations is the necessity
for initiating a timing sequence when blood is applied
to a reagent pad. A user will typically have conducted
a finger stick to a obtain a blood sample and will then
be required to simultaneously apply the blood from the
finger to a reagent pad while initiating a timing
circuit with his or her other hand, thereby requiring
the use of both hands simultaneously. This is
particularly difficult since it is often necessary to
insure that the timing circuit is started only when
blood is applied to the reagent pad. All of the prior
art methods require additional manipulations or
additional circuitry to achieve this result.
Accordingly, simplification of this aspect of
reflectance reading instruments is desirable.
The presence of red blood cells or other coloured
components often interferes with the measurements of
these absolute values thereby calling for exclusion of
red blood cells in these two prior methods as they are
most widely practised. In the device of U.S. patent
3,298,789 an ethyl cellulose membrane prevents red
blood cells from entering the reagent pad. Similarly,
the water—resistant film of U.S. patent 3,630,957
prevents red blood cells from entering. In both cases
the rinse or wipe also acts to remove these potentially
interfering red blood cells prior to measurement.
EP—A—OllOl73 discloses a MBTH—DMAB dry—chemistry porous
strip for determining glucose in whole blood samples
and how that may be used in an instrument such as a
diffuse reflectance spectrophotometer. EP—A—Ol40337
similarly discloses an element and method for detecting
analytes or whole blood by detecting a dye produced
therein spectrophotometrically.
The present invention provides a method for measuring
the concentration of an analyte in whole blood which
comprises:
providing a porous, hydrophilic matrix containing
a signal—producing system which is capable of reacting
with said analyte to produce a light-absorptive dye
product,
said matrix having a first surface, for
receiving an unmeasured sample of said whole blood, and
a second surface, opposite said first surface, to which
at least a portion of said sample can travel through
said matrix; said matrix being such that excess liquid
does not penetrate to said second surface from said
first surface;
applying said sample to said first surface of said
matrix;
allowing at least a portion of said sample to
migrate from said first surface to said second surface;
illuminating said second surface of said matrix
with light of a first wavelength, which can be absorbed
by said light—absorptive dye product, and light of a
second, different wavelength, which can be absorbed by
whole blood;
quantitatively measuring light of said first
wavelength reflected from said second surface of said
matrix after application of said sample without
removing excess sample from said first surface to
provide a sample reading;
quantitatively measuring light of said second
wavelength reflected from said second surface to
provide a background reading for correcting said sample
reading to account for the absorbance of whole blood;
and
calculating a value for the concentration of said
analyte in said sample from said sample and background
readings.
Preferably the method further comprises the step of
quantitatively measuring light reflected from said
second surface prior to application of said sample to
said matrix to provide a baseline reading, wherein said
baseline reading is also used for calculating said
value.
Suitably the present method can be performed employing
a meter for measuring the concentration of an analyte
in whole blood, which is adapted to removably receive a
porous, hydrophilic matrix which contains a signal-
producing system which is capable of reacting with said
analyte to produce a light—absorbtive dye product, said
matrix having a first surface, for receiving an
unmeasured sample of said whole blood, and a second
surface, to which at least
opposite said first surface,
a portion of said sample can travel through said
matrix, said meter comprising:
means for illuminating said second surface of said
matrix with light of a first wavelength, which can be
absorbed by said light—absorbtive dye product, and
light of a second, different wavelength, which can be
absorbed by whole blood;
means for detecting and quantitating the light
reflected from said second surface of said matrix at
said first and second wavelengths;
means for calculating the concentration of said
analyte in said sample based on one reading of the
reflected light of said first wavelength, corrected for
the absorbance of whole blood based on a reading of the
reflected light of said second wavelength; and
means for reporting the calculated concentration
of said analyte.
Suitably the meter further comprises a self—contained
electronic power supply operationally connected to said
means for illuminating, means for detecting, means for
calculating and means for reporting. For measuring the
concentration of glucose in a sample, the said first
wavelength is suitably from 625 to 645 nm (which can be
absorbed by a dye product produced by the reaction of
and said
nm (which
glucose with the signal—producing system)
second wavelength is suitably from 690 to
can be absorbed by whole blood). More suitably, said
first wavelength is about 635 nm and said second
wavelength is about 700 nm.
Preferably the meter further comprises a control means
for causing a timing circuit to be initiated on
detection of a decrease in the amount of light of
either wavelength reflected from said second surface
after application of said sample to said first surface
of said matrix. Suitably, said control means causes one
or more readings of reflected light to be taken at
predetermined intervals after detection of said
decrease. Preferably said control means is capable of
causing said means for detecting to take a reading of
light reflected from said second surface of said matrix
prior to application of said sample to said first
surface of said matrix. More preferably said control
means is further capable of causing said means for
detecting to collect and store a background detector
current reading in the absence of reflected light from
said illuminating means.
Preferably, when power is supplied to said control
means in an analyte detection mode, said control means
automatically causes said means for detecting to read
reflected light, to initiate a timing circuit upon
detection of said decrease, to collect readings at
predetermined intervals after detection of said
decrease, to calculate a value for the concentration of
analyte in said sample from said readings, and to
transfer said value to said reporting means. More
preferably, when power is supplied to said control
means in a baseline reflectance mode prior to an
analyte detection mode, said control means causes said
means for detecting to take a baseline reading of the
reflected light prior to application of said sample to
said matrix and wherein, when said control means is in
said analyte detection mode, said baseline reading is
also used by said calculating means for calculation of
said value.
Suitably the present invention can be carried out using
a kit comprising the presently described meter and a
porous hydrophilic matrix which contains a signal-
producing system which is capable of reacting with said
analyte to produce a light absorbtive dye—product, said
matrix having a first surface, for receiving an
unmeasured sample of said whole blood, and a second
surface, opposite said first surface, to which at least
a portion of said sample can travel through said
matrix.
Preferably each matrix comprises a polyamide. Suitably
the nominal pore size of said matrix is from 0.2 to 1.0
Preferably the kit is for measuring the concentration
of glucose in said whole blood and the signal—producing
system comprises 3—methyl—2-benzothiazolinone hydrazone
hydrochloride and 3-dimethylamino benzoic acid.
Suitably the signal—producing system is at a pH of 3.8
to 5.
The meter may be activated upon a change in reflectance
of the matrix when fluid penetrates the matrix. In use
the sample of whole blood is added to the matrix which
filters out large particles, such as red blood cells,
typically with the matrix present in the meter. The
signal-producing system produces a dye product which
further changes the reflectance of the matrix, which
change can be related to the presence of an analyte in
a sample.
An exemplary diagnostic assay is the determination of
glucose in the whole blood, where the determination is
made without interference from the blood and without a
complicated protocol subject to use error.
The present application is divided from IE 1995/0182.
The present invention can be more readily understood by
reference to the following detailed description when
read in conjunction with the attached drawings,
wherein:
Figure 1 is a perspective view of one embodiment
of a meter suitable for performing the present method
containing the reagent matrix to which the fluid being
analyzed is applied.
Figure 2 is a block diagram schematic of a meter
that can be employed in the practice of the invention.
Figure 3 is a block diagram schematic of an
alternate meter that can be employed in the practice of
the invention.
Detailed Description
The Reagent Matrix
The reagent matrix, its various chemical systems and
its manner of use are described in IE 64442
(1987/2162), from which the above mentioned application
IE 1995/0182 is divided. However, a very brief
description follows with reference to the Figures.
As can be seen from Figure l, a support holds reagent
matrix 11 so that a sample can be applied to one side
of the reagent matrix while light reflectance is
measured from the side of the reagent matrix opposite
the location where sample is applied.
Figure 2 shows a system in which the sample is applied
to the side with the hole in the support while light is
reflected and measured on the other side of the reagent
matrix. Other structures than the one depicted may be
employed. The matrix may take various shapes and forms,
subject to the limitations provided herein. The matrix
will be accessible on at least one surface and usually
two surfaces.
The reagent matrix may be attached to the support by
any convenient means, e.g. a holder, clamp or
adhesives; however, in the preferred mode it is bonded
to the support. The bonding can be done with any non—
reactive adhesive, by a thermal method in which the
support surface is melted enough to entrap some of the
material used for the matrix, or by microwave or
ultrasonic bonding methods which likewise fuse the
matrix to the support. It is important that the bonding
be such as to not itself interfere substantially with
the diffuse reflectance measurements or the reaction
being measured, although this is unlikely to occur as
no adhesive need be present at the location where the
reading is taken. For example, an adhesive 13 can be
applied to the support 12 followed first by punching
hole l4 into the combined support and adhesive and then
applying matrix ll to the adhesive in the vicinity of
hole l4 so that the peripheral portion of the reagent
matrix attaches to the support. The combination of the
reagent matrix and the support is hereafter referred to
as a strip.
When used with whole blood, the reagent matrix
preferably has pores with an average diameter in the
range of from about 0.1 to 2.0 um, preferably about 0.2
to 1.0 and more preferably from about 0.6 to 1.0 um.
A preferred manner of preparing the reagent matrix is
to cast a hydrophilic polymer onto a core of non—woven
fibres. The core fibres can be any fibrous material
that produces the described integrity and strength,
such as polyesters and polyamides. The reagent that
will form the light—absorbing reaction product is
present within the pores of the matrix but does not
block the matrix so that the liquid portion of the
whole blood being analyzed can flow through the pores
of the matrix, while particles, such as erythrocytes,
are held at the surface.
Any signal producing system may be employed that is
capable of reacting with the analyte in the sample to
produce (either directly or indirectly) a compound that
is characteristically absorptive at a wavelength other
than a wavelength at which the whole blood
substantially absorbs, but a reagent producing a light-
absorbtive dye product upon reacting with glucose will
be especially useful.
The Measurement Method
The measurement method relies on a change in
absorbance, as measured by diffuse reflectance, which
is dependent upon the concentration of analyte present
in a sample being tested. This change may be determined
by measuring the change in the absorbance of the test
sample between two or more points in time.
The first step of the method to be considered will be
application of the sample to the matrix. In practice, a
measurement could be carried out as follows: First a
sample of whole blood containing an analyte is
obtained. Blood may be obtained by a finger stick, for
example. An excess over matrix saturation in the area
about 5-10
where reflectance will be measured (i.e.
microliters) of whole blood is applied to the reagent
matrix. Simultaneous starting of a timer is not
required (as is commonly required in the prior art), as
will become clear below. Excess fluid can be removed,
such as by light blotting, but such removal is also not
required. The matrix is typically mounted in a meter
for reading light absorbance; e.g. color intensity, by
reflectance, prior to application of the sample.
Absorbance is measured at certain points in time after
application of the sample. Absorbance refers in this
application not only to light within the visual
wavelength range but also outside the visual wavelength
range, such as infrared and ultraviolet radiation.
From these measurements of absorbance a rate of color
development can be calibrated in terms of analyte
concentration.
The Meter
A suitable instrument, such as a diffuse reflectance
spectrophotometer with appropriate software, can be
made to automatically read reflectance at certain
points in time, calculate rate of reflectance change,
and, using calibration factors, output the
concentration of analyte in the whole blood. Such a
meter is schematically shown in Figure 2 wherein a
reagent matrix of the invention comprising support l2
to which reagent matrix ll is affixed is shown. Light
source 5,
diode
for example a high intensity light emitting
(LED) projects a beam of light onto the reagent
matrix. A substantial portion (at least 25%, preferably
at least 35%, and more preferably at least 50%, in the
absence of reaction product) of this light is
diffusively reflected from the reagent matrix and is
detected by light detector 6, for example a
phototransistor that produces an output current
proportional to the light it receives. Light source 5
and/or detector 6 can be adapted to generate or respond
to a particular wavelength light, if desired. The
output of detector 6 is passed to amplifier 7, for
example, a linear integrated circuit which converts the
phototransistor current to a voltage. The output of
amplifier 7 can be fed to track and hold circuit 8.
This is a combination linear/digital integrated circuit
which tracks or follows the analog voltage from
amplifier 7 and, upon command from microprocessor 20,
locks or holds the voltage at its level at that time.
Analog—to—digital converter 19 takes the analog voltage
from track and hold circuit 8 and converts it to, for
example, a twelve—bit binary digital number upon
command of microprocessor 20. Microprocessor 20 can be
a digital integrated circuit. It serves the following
control functions: 1) timing for the entire system; 2)
reading of the output of analog/digital converter l9;
3) together with program and data memory 21, storing
data corresponding to the reflectance measured at
specified time intervals; 4) calculating analyte
concentrations from the stored reflectances; and 5)
outputing analyte concentration data to display 22.
Memory 21 can be a digital integrated circuit which
stores data and the microprocessor operating program.
Reporting device 22 can take various hard copy and soft
copy forms. Usually it is a visual display, such as a
liquid crystal or LED display, but it can also be a
tape printer, audible signal, or the like. The
instrument also can include a start—stop switch and can
provide an audible or visible time output to indicate
times for applying samples,
taking readings, etc., if
desired.
Reflectance Switching
In the present invention, the reflectance circuit
itself can be used to initiate timing by measuring a
drop in reflectance that occurs when the aqueous
portion of the whole blood applied to the reagent
matrix migrates to the surface at which reflectance is
being measured. Typically, the meter is turned on in a
"ready" mode in which reflectance readings are
automatically made at closely spaced intervals
(typically about 0.2 seconds) from the typically off-
white, substantially dry, The
unreacted reagent matrix.
initial measurement is typically made prior to
penetration of the matrix by fluid being analyzed but
can be made after the fluid has been applied to a
location on the reagent matrix other than where
reflectance is being measured. The reflectance value is
evaluated by the microprocessor, typically by storing
successive values in memory and then comparing each
value with the initial unreacted value. When the
aqueous portion penetrates the reagent matrix, the drop
in reflectance signals the start of the measuring time
interval. Drops in reflectance of 5—50% can be used to
initiate timing, typically a drop of about 10%. In this
simple way there is exact synchronization of aqueous
portion reaching the surface from which measurements
are taken and initiation of the sequence of readings,
with no requirement of activity by the user.
Although the total systems described in this
application are particularly directed to the use of
polyamide matrices and particularly to the use of such
matrices in determining the concentration of various
sugars, such as glucose, and other materials of
biological origin, there is no need to limit the
reflectance switching aspect of the invention to such
matrices. the matrix used with reflectance
For example,
switching may be formed from any water—insoluble
hydrophilic material and any other type of reflectance
assay.
Particular Application to Glucose Assay
A particular example with regard to measuring the
concentration of glucose in the presence of red blood
cells will now be given in order that greater detail
and particular advantage can be pointed out. Although
this represents a preferred embodiment of the present
the invention is not limited to the
invention,
detection of glucose in blood.
The use of polyamide surfaces to form the reagent
matrix provides a number of desirable characteristics.
These are that the reagent matrix is hydrophilic (i.e.
takes up reagent and sample readily), does not deform
on wetting (so as to provide a flat surface for
reflectance reading), is compatible with enzymes (in
order to impart good shelf stability), takes up a
limited sample volume per unit volume of matrix
(necessary in order to demonstrate an extended dynamic
range of measurements), and shows sufficient wet
strength to allow for routine manufacture.
._18_
In a typical configuration, the method is carried out
using a meter consisting of a plastic holder and the
reagent matrix having the signal producing system
impregnated therein, as described in IE 64442.
The meter used to make the reflectance readings
minimally contains a light source, a reflected light
detector, an amplifier, an analog to digital converter,
a microprocessor with memory and program, and a display
device.
The light source typically consists of a light emitting
diode (LED). Although it is possible to use a
polychromic light source and a light detector capable
of measuring at two different wavelengths, a preferred
meter would contain two LED sources or a single diode
capable of emitting two distinct wavelengths of light.
Commercially available LEDs producing the wavelengths
of light described as being preferred in the present
specification include a Hewlett Packard HLMP—l34O with
an emission maximum at 635 nm and a Hewlett Packard
QEMT—lO45 with a narrow—band emission maximum at 700
nm. Suitable commercially available light detectors
include a Hammamatsu 5874—l8K and a Litronix BPX—65.
Although other methods of taking measurements are
feasible, the following method has provided the desired
results. Readings are taken by the photodetector at
specified intervals after timing is initiated. The 635
nm LED is powered only during a brief measuring time
span that begins approximately 20 seconds after the
-19..
start time as indicated by reflectance switching. If
this reading indicates that a high level of glucose is
present in the sample, a 30—second reading is taken and
used in the final calculation in order to improve
accuracy. Typically, high levels are considered to
begin at about 250 mg/dl. The background is corrected
with a 700 nm reading taken about l5 seconds after the
start of the measurement period. The reading from the
photodetector is integrated over the interval while the
appropriate LED is activated, which is typically less
than one second. The raw reflectance readings are then
used for calculations performed by the microprocessor
after the signal has been amplified and converted to a
digital signal. Numerous microprocessors can be used to
carry out the calculation. An AIM65 single—board
microcomputer manufactured by Rockwell International
has proven to be satisfactory.
The present method allows a very simple procedure with
minimum operational steps on the part of the user. In
use, a strip is placed in the detector so that the hole
in the strip registers with the optics of the meter. A
removable cap or other cover is placed over the optics
and strip to shield the assembly from ambient light.
The measurement sequence is then initiated by pressing
a button on the meter that activates the microcomputer
to take a measurement of reflected light from the
unreacted reagent matrix, called an Rwy reading. The
cap is then removed and a drop of blood is applied to
the reagent matrix, typically while the reagent matrix
is registered with the optics and the meter. It is
preferred that the strip be left in register with the
optics in order to minimize handling. The meter is
capable of sensing the application of blood by a
decrease in the reflectance when the sample passes
through the matrix and reflected light is measured on
the opposite side. The decrease in reflectance
initiates a timing sequence which is described in
detail at other locations in this specification. The
cover should be replaced within 15 seconds of sample
application, although this time may vary depending on
the type of sample being measured. Results are
typically displayed at approximately 30 seconds after
blood application when a blood glucose sample is being
measured, although a 20 second reaction is permissible
for blood samples having a concentration of glucose of
less than 250 mg/dl.
A particularly accurate evaluation of glucose
concentration
(or any other analyte being measured) can
be made using the background current, i.e. the current
from the photodetector with power on but with no light
reflected from the reagent matrix, in order to make a
background correction. It has been demonstrated that
over a 2-3 month period that this value does not change
for a particular meter, and it is possible to program
this background reading into the computer memory as a
constant. With a slight modification of the procedure,
however, this value can be measured with each analysis
for more accurate results. In the modified procedure
the meter would be turned on with the lid closed before
the strip is in place, and the background current would
be measured. The strip would then be inserted into the
meter with the cover closed, an Rwy measurement taken,
and the procedure continued as described above. With
this modified procedure the background current does not
need to be stable throughout the life of the meter,
thereby providing more accurate results.
The raw data necessary for calculating a result in a
glucose assay are a background current reported as
background reflectance, Rb, as described above; a
reading of the unreacted matrix, also described
Rdry I
above; and an endpoint measurement. Using the preferred
embodiments described herein, the endpoint is not
particularly stable and must be precisely timed from
the initial application of blood. However, the meter as
described herein performs this timing automatically.
For glucose concentrations below 250 mg/dl, a suitably
stable endpoint is reached in 20 seconds, and a final
reflectance, Rm, is taken. For glucose concentrations
up to 450 mg/dl, a 30—second reflectance reading, Rgm
is adequate. Although the system described herein
displays good differentiation up to 800 mg/dl of
glucose, the measurement is somewhat noisy and
inaccurate above 450 mg/dl, although not so great as to
cause a significant problem. Longer reaction times
should provide more suitable readings for the higher
levels of glucose concentration.
The 700 nm reflectance reading for the dual wavelength
(R15)- BY
this time blood will have completely saturated the
measurement is typically taken at l5 seconds
reagent matrix. Beyond 15 seconds the dye reaction
continues to take place and is sensed, to a small part,
by a 700 nm reading. Accordingly, since dye absorption
by the 700 nm signal is a disadvantage, readings beyond
seconds are ignored in the calculations.
The raw data described above are used to calculate
parameters proportional to glucose concentration which
can be more easily visualized than reflectance
measurements. A logarithmic transformation of
reflectance analogous to the relationship between
absorbtivity and analyte concentration observed in
transmission spectroscopy (Beer's Law) can be used if
desired. A simplification of the Kubelka—Monk
equations, derived specifically for reflectance
spectroscopy, have proven particularly useful. In this
derivation K/S is related to analyte concentration with
K/S defined by Equation 1.
K/S—t = (1 — R*t)2/(2 x R*t) (1)
R*t is the reflectivity taken at a particular endpoint
time, t, and is the absorbed fraction of the incident
light beam described by Equation 2, where Rt is the
endpoint reflectance,
R20 OI R30 .
R*t = (Rt ‘ Rb)/(Rdry ‘ Rb) (2)
R*t varies from O for no reflected light (Rb) to 1 for
(Rdry> -
the calculations greatly simplifies meter design as a
total reflected light The use of reflectivity in
highly stable source and a detection circuit become
unnecessary since these components are monitored with
each Rwy and Rb measurement.
For a single wavelength reading K/S can be calculated
at 20 seconds (K/S-20) or 30 seconds (K/S—30). The
calibration curves relating these parameters to YSI
(Yellow Springs Instruments) glucose measurements can
be precisely described by the third order polynomial
equation outlined in Equation 3.
YSI = a0 + a1 (K/S) + a2 (K/S)2 + a3 (K/s)3 (3)
The coefficients for these polynomials are listed in
Table 1.
TABLE 1
Coefficients for Third Order Polynomial Fit of Single
Wavelength Calibration Curves
K/S—2O K/S-30
ao -55.75 -55.25
a; 0.1632 0.1334
a2 -5.765 x 10* -2.241 x 10”
a3 2.58 x 10* 1.20 x 10“
The single chemical species being measured in the
preferred embodiments is the MBTH—DMAD indamine dye and
the complex matrix being analyzed is whole blood
distributed on a O.8u Posidyne membrane.
The use of the MBTH—DMAB couple allows for correction
of hematocrit and degree of oxygenation of blood with a
single correction factor. The more typically used
bengidine dyes do not permit such a correction. The dye
forms a chromophore that absorbs at approximately 635
nm but not to any significant extent at 700 nm. Slight
variations in measuring wavelengths (+/— about 10 nm)
are permitted. At 700 nm both hematocrit and degree of
oxygenation can be measured by measuring blood colour.
A review entitled "Application of Near Infra Red
Spectrophotometry to the Nondestructive Analysis of
Foods A Review of Experimental Results", CRC Critical
Reviews in Food Science and Nutrition, 18(3) 203-30
(1983), describes the use of instruments based on the
measurement of an optical density difference AOD (Aa-
Ab) where ODAa is the optical density of the wavelength
corresponding to the absorption maximum of a component
to be determined and ODAb is the optical density at a
wavelength where the same component does not absorb
significantly.
The algorithm for dual wavelength measurement is by
necessity more complex than for single wavelength
measurement but is much more powerful. The first order
correction applied by the 700 nm reading is a simple
subtraction of background color due to blood. In order
to make this correction, a relationship between
absorbance at 635 nm and 700 nm due to blood color can
be and was determined by measuring blood samples with O
mg/dl glucose over a wide range of blood color. The
color range was constructed by varying hematocrit, and
fairly linear relationships were observed. From these
lines the K/S—l5 at 700 nm was normalized to give
equivalence to the K/S—3O at 635 nm. This relationship
is reported in Equation 4, where K/S—l5n is the
normalized K/S-l5 at 700 nm.
K/S—l5n = (K/S—15 x 1.54) — 0.133 (4)
Note that the equivalence of the normalized 700 nm
signal and the 635 nm signal is only true at zero
glucose. The expressions from which the calibration
curves were derived are defined by Equations 5 and 6.
K/S—20/15 (K/8-20) -
(K/S—l5n) (5)
K/S—30/l5 (K/S—30) — (K/S—l5n) (6)
These curves are best fit by fourth—order polynomial
equations similar to Equation 3 to which a fourth—order
term in K/S is added. The computer—fit coefficients for
these equations are listed in Table 2.
TABLE 2
Coefficients for Fourth—Order Polynomial Fit of Dual
Wavelength Calibration Curves
K/S—20/l5 K/s—30/15
ag —0.1388 1.099
a1 0.1064 0.05235
a2 6.259 x 10“ 1.229 x 10"‘
a3 -6.12 x 10*“ -5.83 x 10*“
a4 3.21 x 1011 1.30 x 10“
A second order correction to eliminate errors due to
chromatography effects has also been developed. Low
hematocrit samples have characteristically low 700 nm
readings compared to higher hematocrit samples with the
(K/S—30)/(K/S-
) is plotted versus K/S—3O over a wide range of
same 635 nm reading. When the ratio of
hematocrits and glucose concentrations, the resulting
line on the graph indicates the border between samples
which display chromatography effects (above the curve)
and those that do not
(below the curve). The K/S—3O for
the samples above the curve are corrected by elevating
the reading to correspond to a point on the curve with
the same (K/S—30)/(K/S—l5).
The correction factors reported above were tailor made
to fit a single meter and a limited number of reagent
preparations. The algorithm can be optimized for an
individual meter and reagent in the same manner that is
described above.
In summary, the system of the present invention
minimizes operator actions and provides numerous
advantages over prior art reflectance-reading methods.
When compared to prior methods for determining glucose
in blood, for example, there are several apparent
advantages. First, the amount of sample required to
saturate the thin reagent matrix is small (typically 5-
microliters). Second, operator time required is only
that necessary to apply the sample to the thin reagent
matrix and close the cover (typically 4-7 seconds).
Third, no simultaneous timing start is required.
Fourth, whole blood can be used. The method does not
require any separation or utilization of red—cell-free
samples.
The invention now being generally described, the same
will be better understood by reference to the following
specific examples which are presented for purposes of
illustration only and are not to be considered limiting
of the invention unless so specified.
Example 1
Reproducibility:
One male blood sample (JG, hematocrit = 45) was used to
collect the reproducibility data set forth in Tables 3-
TABLE 3
Reproducibility of a Single Wavelength MPX System
YSI Average (mg/dl) S.D. (mg/dl) %C.V.
(mg/dl) 20 sec. 30 sec. 20 sec. 30 sec. 20 sec. 30 sec.
23.1 23.0 2.1 2.04 9.1 9.0
55 53.3 53.2 3.19 3.32 6.0 6.3
101 101 101 3.0 3.3 3.0 3.3
326 326.6 327 13.3 9.8 4.1 3.0
501 503 17.1 3.4
690 675 28 4.15
810 813 37 4.5
TABLE 4
Reproducibility of a Dual Wavelength MPX System
YSI Average (mg/dl) S.D. (mg/dl) %C.V.
(mg/dl) 20 sec. 30 sec. 20 sec. 30 sec. 20 sec. 30 sec.
25 27 1.34 1.55 5.4 5.7
55 55 57.4 2.58 2.62 4 7 4.6
101 101 101.5 2.55 2.18 2.5 2.1
326 332 330 15.0 7.1 4 5 2.1
501 505 21.3 4.2
690 687 22.8 3.3
810 817 30.4 3.7
TABLE 5
Reproducibility of a 3.0mm Diameter Aperture
YSI (mg/dl) % C.V.
.7 mm 3.0 mm
55-100 4.8 4.9
300 3.0 5.0
600 3.8 5.5
avg. 3.9 5.1
The blood was divided into aliquots and spiked with
glucose across a range of 25-800 mg/dl. Twenty
determinations were made at each glucose test
concentration from strips taken at random from a 500
strip sample (Lot FJ4—49B). The results of this study
lead to the following conclusions:
. Single vs. Dual Wavelength: The average C.V. for the
—second dual result was 3.7% vs. 4.8% for the 30-
second single wavelength result, an improvement of 23%
across a glucose range of 25-810 mg/dl. There was a 33%
improvement in C.V. in the 25-326 mg/dl glucose range.
Here the C.V. decreased from 5.4% to 3.6%, a
significant improvement in the most used range. The 20-
second dual wavelength measurement gave similar
improvements in C.V. compared to the single wavelength
measurement in the 25-325 mg/dl range (Tables 3 and 4).
2. Dual Wavelength, 20 vs.
—second Result: The
average C.V. for a 20—second result in the 25-100 mg/dl
range is nearly identical to the 30—second reading,
4.2% vs. 4.1%.
However, at 326 mg/dl the 30—second
reading has a C.V. of 2.1% and the 20—second result a
C.V. of 4.5%. As was seen in the K/S—20 response curve,
the slope begins to decrease sharply above 250 mg/dl.
This leads to poor reproducibility at glucose
concentrations greater than 300 mg/dl for the 20—second
result. From this reproducibility data the cut—off for
the 20—second result is somewhere between 100 and 326
mg/dl. A cut—off of 250 mg/dl was later determined from
the results of the recovery study set forth in Example
. Aperture Size: A smaller optics aperture size, 3.0
mm vs. 5.0 mm., was investigated. Initial
experimentation using a 10- replicate, hand—dipped disk
sample did show improved C.V.s with the 3.0mm aperture,
apparently because of easier registration with the
when machine—made roll membrane
system optics. However,
was used, the average C.V. (Table 5) of the larger
aperture, 4.7mm, was 3.9% vs. an average C.V. for the
.0mm aperture of 5.1%. This 30% increase in C.V. was
probably due to the uneven surface of the roll membrane
lot as discussed below.
Example II
Recovery:
For comparison of the present method (MPX) against a
typical prior art method using a Yellow Springs
Instrument Model 23A glucose analyser manufactured by
Yellow Springs Instrument Co., Yellow Springs, Ohio
(YSI), blood from 36 donors was tested. The donors were
divided equally between males and females and ranged in
hematocrit from 35 to 55%. The blood samples were used
within 30 hours of collection, with lithium heparin as
the anti—coagulant. Each blood sample was divided into
aliquots and spiked with glucose to give 152 samples in
the range of 0-700 mg/dl glucose. Each sample was
tested in duplicate for a total of 304 data points.
Response curves were constructed from these data and
glucose values then calculated from the appropriate
(Tables 1 and 2).
equation These MPX glucose values
were then plotted vs. the YSI values to give
scattergrams.
Comparison of MPX Systems: For both the 20—second and
—second measurement times there is visually more
scatter in the single—wavelength scattergrams than the
dual—wavelength scattergrams. The 20—second reading
becomes very scattered above 250 mg/dl but the 30-
second measurement does not have wide scatter until the
glucose level is ;500 mg/dl.
These scattergrams were quantitated by determining the
deviations from YSI at various glucose concentrations.
The following results were obtained.
TABLE 6
Accuracy of MPX from Recovery Data
MPX Measurement (ggfgl) C_V_ for Range+
Wwelength Time (5904 0~50 50-250 250-450 450-70
Single 20 ir5.6 7.2 14.5 —
Single 30 :59 7.1 8.8 10.2
Dual 20 i2.3 5.3 12.8 —
Dual 30 :2.l9 5.5 5.8 8.4
ggtg: These are inter method C.V.s.
a. The dual wavelength system gave C.V.s that ranged 30% lower
than the single wavelength system.
b. The single wavelength system,
from 0-50 mg/dl, showed a S.D. of
i6—7 mg/dl whereas the S.D. for a dual wavelength measurement was
only i2.2 mg/dl.
c. The cut—off for a 30—second MPX measurement is 250 mg/dl. For
the 50-250 mg/dl range both the 20- and 30—second measurements
gave similar inter—method C.V.s (approximately 7% for single
wavelength, 5.5% for dual wavelength). However, in the 250-450
mg/dl range inter—method C.V.s more than double for the 20-second
reading to 14.5% for the single and l2.8% for the dual wavelength.
d. The 30-second reading was unusable above 450 mg/dl for both the
single and dual wavelength measurement (C.V.s of 10.2 and 8.4%).
In conclusion, two MPX systems gave optimum
quantitation in the 0-450 mg/dl range.
. MPX 30 Dual: This dual wavelength system gave a
—second measurement time with a 95% confidence limit
(defined as the probability of a measurement being
of 11.3%
(Table 7)
within 2 S.D. of the YSI) (C.V.) for the range
from 50-450 mg/dl
O-50 mg/dl.
and i4.4 mg/dl (S.D.) for
. MPX 30/20 Dual: This dual wavelength system
gave a 20-second measurement time in the 0-250 mg/dl
range and a 30-second time for the 250-450 range. The
95% confidence limits are nearly identical to the MPX
11.1%
for 0-50 mg/dl).
Dual System (Table 7), (C.V.) for 50-450 mg/dl
and $4.6 mg/dl (S.D.
TABLE 7
Comparison of 95% Confidence Limits for MPX, Glucoscan Plus
and Accu-Chek bG* Reagent Strips
Measuring
Range mg/dl MPX Single Wavelength MPX Dual Wavelength
sec. 30 sec. 20 sec. 30 sec.
0-50 11.2 mg/dl 13.8 mg/dl 4.6 mg/dl 4.4 mg/dl
50-250 14.4% 14.2% 10.6% 11.0%
250-450 - 17.6% — 11.6%
77‘405 Glucoscan Plus (Drexler Clinical) 15-9%
77”405 Accu-Chek bG (Drexler Clinical) 10-7%
50-450 MPX 20/30 Dual Hybrid 11-1%
50‘450 MPX 30 Dual 11-3
* Confidence limits for MPX were from the YSI. The confidence
limits for Glucoscan Plus and Accu-Chek bG were from the
regression equation vs.
differences in calibration.
YSI which eliminates bias due to small
Example III
Stability:
Most of the bench—scale work carried out in optimizing
stability was completed using hand—dipped O.8u Posidyne
membrane disks. The specific dye/enzyme formulation was
set forth previously.
l. Room Temperature Stability: This study attempted to
chart any change in response of the O.8u Posidyne
membrane reagent stored at l8—20°C over silica gel
desiccant. After 2.5 months there was no noticeable
change as measured by the response of a room
temperature sample vs. the response of a sample stored
at 5°C. Each scattergram represented a glucose range of
-450 mg/dl.
. Stability at 37°C: A 37°C stability study using the
same reagent as the RT study was carried out. The
differences in glucose values of reagent stressed at
37°C vs. RT reagent, for matrices stressed with and
without adhesive, was plotted over time. Although the
data was noisy, due to the poor reproducibility of
handmade strips, the stability was excellent for strips
whether they were stressed with or without adhesive.
. Stability at 56°C: Eight 5- to 6-day stability
studies were carried out using different preparations
of a similar formulation on disk membrane (Table 8).
For the low glucose test level (80-lOO mg/dl) the
average glucose value dropped upon stressing by 3.4%,
with the highest drop being 9.55%.
At the high test
level (280-320 mg/dl) the glucose reading declined by
an average of 3.4%, the largest decline being 10.0%.
Stability of pH =
.8 Posidyne Disk Reagent Formulation
Stressed for 5 to 6 Days at 56°C
Difference (56°C vs. RT Sample)
Average of 8 -3.4 -3.4
* These two samples contained twice the normal concentration
of enzyme and dye.
A study of the 56°C stressing of this matrix over a 19-
day period showed no major difference for strips
stressed with or without adhesive.
In both cases the
-day decline in glucose value was
levels (80-100) and 300 mg/dl. Another 56°C study using
hand-dipped 0.8u Posidyne membrane with twice the
normal concentration of enzyme and dye was completed.
Two separate preparations of the same formulation were
made up and the stability measured over a 14-day
period. The average results of the two studies were
plotted. Changes were within ilO% over the 14-day
period at both the high and low glucose test level.
These data show this formulation to be particularly
stable.
Example IV
Sample Size:
The sample size requirements for MPX are demonstrated
in Table 9.
TABLE 9
Effect of Sample Size on MPX Measurements
Sample Size Dual Wavelength Single Wavelength
(pl) Average Average
Low Glucose YSI = 56
3 41 50 39 31 40 31 42 30 19 30
4 44 49 49 49 48 4l 45 44 45 44
54 48 49 51 50 50 49 48 49 49
l0 48 48 50 47 48 54 53 56 55 54
49 49 50 49 55 57 58 60 58
High Glucose YSI = 360
260 276 286 280 274 232 244 260 252
4 383 378 367 341 367 361 356 342 318 344
398 402 382 370 388 378 387 366 35l 370
364 362 378 368 368 356 358 379 369 366
375 370 380 378 376 380 382 389 385 384
The volumes reported in the table were transferred to
the reagent matrix shown in Figure l using a micro
pipet. When blood from a finger stick is applied to a
strip the total sample cannot be transferred, therefore
the volumes reported here do not represent the total
sample size needed to be squeezed from the finger for
the analysis. A 3-ul sample is the minimum necessary to
completely cover the reagent matrix circle. This does
not provide enough sample to completely saturate the
reagent matrix and MPX gives low results. A 4—ul sample
barely saturates the reagent matrix, while a 5—ul
sample is clearly adequate. A lO—ul sample is a large
shiny drop and a 20—ul sample is a very large drop and
is only likely to be used when blood from a pipet is
used for sampling.
At low glucose concentration the single wavelength
result has some dependence on sample size which is
completely eliminated using the dual wavelength
measurement. Although this dependence with the single
wavelength might be considered acceptable, it is
clearly undesirable.
Example V
Reproducibility:
Experimental measurements described above were always
run in replicate, usually 2, 3 or 4 determinations per
data point. These sets have always shown close
agreement even for samples with extreme hematocrits or
extreme oxygen levels. C.V.s were well below 5%. It
appears, therefore, that reproducibility is very good
to excellent.
The subject invention provides for many advantages over
systems which are presently available commercially or
have been described in the literature. The protocols
are simple and require little technical skill and are
relatively free of operator error. The assays can be
carried out rapidly and use inexpensive and relatively
harmless reagents, important considerations for
materials employed in the home. The user obtains
results which can be understood and used in conjunction
with maintenance therapy. In addition, the reagents
have long shelf lives, so that the results obtained
will be reliable for long periods of time. The meter
which has been described and can be employed in the
present method is simple and reliable and substantially
automatic.
The invention now being fully described, it will be
apparent to one of ordinary skill in the art that many
modifications and changes can be made thereto.
Claims (4)
- l. A method for measuring the concentration of an analyte in whole blood which comprises: providing a porous, hydrophilic matrix containing a signal—producing system which is capable of reacting with said analyte to produce a light—absorptive dye product, said matrix having a first surface (15), for receiving an unmeasured sample of said whole blood, and a second surface (16), opposite said first surface (15), to which at least a portion of said sample can travel through said matrix; said matrix being such that excess liquid does not penetrate to said second surface (16) from said first surface (15); applying said sample to said first surface (15) of said matrix; allowing at least a portion of said sample to (15) migrate from said first surface to said second surface (16); illuminating said second surface (16) of said matrix with light of a first wavelength, which can be absorbed by said light—absorptive dye product, and light of a second wavelength, which can be absorbed by whole blood; quantitatively measuring light of said first wavelength reflected from said second surface (l6) of said matrix after application of said sample without removing excess sample from said first surface (15) to produce a sample reading; quantitatively measuring light of said second wavelength reflected from said second surface (l6) to provide a background reading for correcting said sample reading to account for the absorbance of whole blood; and calculating a value for the concentration of said analyte in said sample from said sample and background readings.
- 2. The method of claim 1, which further comprises the step of quantitatively measuring light reflected from said second surface prior (l6) to application of said sample to said matrix to provide a baseline reading, wherein said baseline reading is also used to calculate said value.
- 3. The method of claiH1 1 or 2 wherein the matrix comprises a polyamide.
- 4. The method of claim 3, wherein the nominal pore size of said matrix is from 0.2 to 1.0 um. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
USUNITEDSTATESOFAMERICA13/08/19868 | |||
US06/896,418 US4935346A (en) | 1986-08-13 | 1986-08-13 | Minimum procedure system for the determination of analytes |
Publications (2)
Publication Number | Publication Date |
---|---|
IE20020900A1 IE20020900A1 (en) | 2003-07-23 |
IE83676B1 true IE83676B1 (en) | 2004-11-17 |
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