Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ROCHE DIAGNOSTICS GMBH 532210B/W0
Electrochemical sensor for determining blood clotting, corresponding system
for
measuring blood clotting and method for determining blood clotting
The invention concerns a sensor based on dry chemistry for the determination
of
blood coagulation which has at least 2 electrodes on an inert support and a
dry
reagent. The invention additionally concerns a blood coagulation measuring
system
containing an electrochemical sensor and an instrument for measuring
electrical
current. Finally the invention also concerns a method for determining blood
coagulation by means of an electrochemical sensor.
EP-B 0 441 222 describes a method and sensor electrode system for the
electrochemical determination of an analyte or an oxidoreductase. The patent
document discloses the role of a reducible substance as an electron carrier in
a
redox reaction of an analyte to be determined on an electrode. A typical
analyte is
glucose, lactate or a redox enzyme such as glucose dehydrogenase or lactate
dehydrogenase.
An electrochemical method for determining proteases and antiproteases is known
from EP-B 0 018 002. This uses a protease or antiprotease substrate composed
of an
oligopeptide to which an aromatic or heterocyclic amine or polyamine is bound.
The enzyme to be determined cleaves the bond between the carboxyterminal amino
acid and the amine or polyamine and the amount of released amine or polyamine
is
determined electrochemically. The determination of freely mobile components of
the blood coagulation cascade in solution is described.
An electrochemical sensor for coagulation detection is known from C. Bisson et
al.,
"A microanalytical device for the assessment of coagulation parameters in
whole
blood", Technical Digest Solid-State Sensor and Actuator Workshop, Hilton Head
Island, South Carolina, USA, June 8-11, 1998. This publication describes in
general
the possibility of using electrochemical coagulation sensors with amperometric
detection. Details of the substrates used are for example not disclosed.
Furthermore
CA 02400651 2002-08-19
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the described electrodes and test devices are complicated to manufacture and
are
thus unattractive for a mass market.
In general it can be stated that the prior art does not provide a simple and
reliable
starting point for electrochemical coagulation sensors and corresponding
methods
for coagulation detection.
In order to resolve this problem the present invention provides an
electrochemical
sensor, a measuring system for blood coagulation and a method for determining
blood coagulation as described in the independent patent claims. Preferred
embodiments are given in the dependent claims.
The sensor according to the invention is an analytical element based on dry
chemistry. The sensor contains at least two electrodes of which at least one
electrode
is a so-called working electrode. In a preferred embodiment it contains no
classical
reference electrode such as an Ag/AgCI reference electrode, but only at least
one
working electrode and one counterelectrode. The electrodes can be composed of
all
conventional electrode materials such as metals, noble metals, alloys or
graphite and
are preferably composed of noble metals such as gold or palladium, or
graphite. The
various electrodes of the sensor can be composed of the same or different
materials.
In a particularly preferred embodiment the sensor contains a working electrode
and
a counterelectrode that are both made of palladium.
The dry reagent of the sensor according to the invention contains for example
the
compound (I)
HZN ~ ~ NH-CO-Arg-Pro-Gly-Tos (I)
as a thrombin substrate. Arg denotes arginine, Pro denotes proline and Gly
denotes
glycine; Tos corresponds to the amino protecting group tosyl. In general any
residues that can be cleaved by thrombin can be used as peptide residues of
the
CA 02400651 2002-08-19
thrombin substrate according to the invention such that phenylenediamine that
can
be determined electrochemically is released from the substrate.
The enzyme thrombin is the Iast protease of the coagulation cascade and is
only
formed during blood coagulation from the protein prothrombin. Hence it is
possible to monitor the clotting of blood and thus determine clotting time by
measuring the enzyme activity of thrombin.
When the thrombin substrate is cleaved, a p-aminoaniline (phenylenediamine) is
farmed which is oxidized on the working electrode of the sensor as described
in
EP-B 0 441 222. The released electrons are detected. Basically all the
compounds
which are described as electron carriers (or mediators) of type 2 in EP-B 0
441 222
can in principle be used as the electrochemically detectable part of the
thrombin
substrate according to the invention.
In the present invention it has turned out to be particularly advantageous to
add the
reagent glucose-dye-oxidoreductase (GIucDOR) to the reagent as an
amplification
system which oxidizes the glucose present in the blood to be examined and in
this
process uses the type 2 electron carrier released by the thrombin activity as
an
electron acceptor.
The reaction scheme is as follows:
thrombin ' glucono-
peptide-NH: ~ ~ NHZ HEN ~ ~ NHz lactone
peptide
electrode ~~ 2e~ ~R
m
HN ~ NHZ ~ glucose
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The primary oxidation product of the type 2 electron carrier is a quinone
diirnine
which can be recycled in the form of p-aminoaniline by means of a dye oxido-
reductase such as glucose-dye oxidoreductase (GIucDOR) and can thus again
transfer electrons to the working electrode. Hence it is possible to
considerably
amplify the original signal. In addition to GIucDOR, there are other enzymes
(such
as alcohol dehydrogenase or oxidases such as glucose oxidase or lactate
oxidase)
which can convert the quinone diimine into phenylenediamine. Additional
special
substrates (corresponding to the glucose for GIucDOR) are required for this
(such
as alcohols for the enzyme alcohol dehydrogenase or lactate for lactate
oxidase)
which can then be optionally incorporated in suitable amounts into the reagent
formulation.
The electrochemical determination ofblood coagulation occurs quasi-
potentiostatically, preferably using a 2 electrode system in which one
electrode
operates simultaneously as a reference and counterelectrode and the other
electrode
operates as a working electrode. A constant voltage is applied to this 2
electrode
system and the current is measured over time. This method is also referred to
as an
amperometric measurement procedure. In order to determine the blood clotting
time, the current time course is measured and it is determined after which
time
period from the start of the coagulation measurement the measured current
exceeds
a predetermined threshold value. This time period is a measure for the
clotting
time.
In addition to the amperometric measuring methods, it is also possible to use
voltammetric measuring methods. In this case the voltage between the
electrodes is
not set to be constant, but rather the voltage is changed linearly from an
initial value
to a final value and subsequently returned to the initial value. This process
can be
repeated several times aver the entire measurement period.
In the voltammetric method the current is plotted against the voltage and then
when this is repeated one obtains a nested set of current-voltage curves
(cyclovoltammograms). If the voltage range is suitable, oxidation peaks and
reduction peaks of the electron carrier are formed in these curves. The height
of
these peaks is directly proportional to the concentration of the electron
carrier
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S
provided that other redox-active substances are not co-oxidized or co-reduced
in
the covered potential range and thus additionally contribute to the current.
Such an
interference may be ignored when measuring a concentration change.
If the current values for the peak maxima or the areas enclosed by the curves
(corresponding to the charge turnover) of the individual current voltage loops
are
plotted against time, one also obtains a picture of the concentration change
of the
electron carrier over the measurement period in a time raster given by the
cycle
period. This information can then be used - as in the amperometric methods -
to
determine the blood clotting time.
Oligopeptide derivatives of the following general formula (II) are used for a
coagulation test which is based on the determination of the enzyme activity of
a
protease e.g. thrombin:
O
H
X1/'X2wX~ N Xa~~X5
(II)
NH
H
2N NH
In this case X,, X2 and X3 represent natural or artificial amino acids
including
possible protective groups. The sequence X,-Xz-X3 is selected according to the
desired application, for example for detection by means of thrombin, and has
been
described for various purposes in numerous publications (c~ e.g. U. Becker et
al.,
Clin. Chem., 30 (4), 524-S28 (1984); M.-C. Bouton et al., Eur. J. Biochem.,
229 (2),
526-532 ( 1995); H.D. Bruhn et al., "Hamostaseologie", 15 ( 1 ), 54-S6 (
1995); E. De
Candia et al., Thromb. Haemostasis, 77 (4), 735-740 (1997) ; P.L. Coleman et
al.,
Clin. Chem., 29 (4), 603-608 (1983); J. DiMaio et al., J. Med. Chem., 35, 3331-
3341
( 1992); A. Frigola et al., J. Clin. Pathol., 32 ( 1 ), 21-25 ( I979); P. J.
Gaffney et al.,
Thromb. Res., 10 (4), 549-556 (I977); J. Hofsteenge et aL, Biochem. J., 237,
243-251
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(1986); R. Lottenberg et al., Thromb. Res., 28, 3I3-332 (1982); M.K. Ramjee,
Anal.
Biochem., 277, 11-18 (2000); J.). Slon-Usakiewicz et al., Biochern. 36, 13494-
13502
( 199?); S.A. Sonder et al., Clin. Chem. 32 (6), 934-937 ( 1986); C. Soria et
al.,
Thromb. Res., 19 (3), 435-440 (I980); T. Steinmetzer et al., J. Med. Chem.,
42,
3109-3115 (1999); A. Tripodi et al., Clin. Chem., 30 (8), 1392-1395 (1984); J.
I.
Wining et al., Thromb. Res., 46 (4), 567-574 (1987); EP-B 0 018 002; US
5,108,890;
US 5,190,862; EP-A 0 280 610; EP-B 0 144 744). Furthermore some of these amino
acid sequences are also offered commercially by various companies to detect
different coagulation factors for example from Pentapharrn Ltd, Basle,
Switzerland
or from Chromogenix Germany, Haemochrom Diagnostica GmbH, Essen,
Germany.
A moiety X4-X5 is attached as a leaving group to the amino acid sequence XI-XZ-
X3
and is cleaved by the proteolytic activity of the coagulation factor i.e.
thrombin for
example. Depending on the desired detection principle, the moiety X4-XS that
is
used to detect the proteolytic activity is coloured (photametric
determination),
fluorescent (cf. inter alia M.K. Ramjee, Anal. Biochem., 277, 11-18 (2000)) or
electroactive (cf. EP-B 0 018 002).
The moiety X4-XS is electroactive within the scope of the present invention
i.e. can
be converted at an electrode with release or uptake of electrons. XQ-XS
preferably
has a maximum oxidation potential of 350mV relative to an AgIAgCI electrode
and
can be detected by an electrochemical measurement.
In this connection X4 can be an atom or group which forms the link with the
oligopeptide sequence N or NH.
The moiety XS can have the following structure (III)
R3
__
N R2 (III)
Ra
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in which R, and RZ are independently of one another alkyl, hydroxyalkyl,
alkoxy-
alkyl, phosphoramide alkyl, polyhydroxyalkyl, sulfone alkyl or hydrogen and R3
and
R4 are OH, O-alkyl, alkyl, H, halogen, NR1R2, COZR, CONR1R2, SRS, NRi-CO-Rl,
O-CO-R,, H.
Peptide substrates of this type in which X4 = N or NH are for example
described in
the following patents or patent applications: JP-A 56042597; JP-A 58090535;
WO-A 86/01209; US 5,059, 525; JP-A 61112096; JP-A 03157353.
XS can alternatively have the following structure (IV):
(IV)
R3
in which R, to R3 have the meaning given above for formula III.
Peptide substrates of this type in which X4 = NR are known (c~ for example JP-
A
03271299; EP-B 0 350 915; P. Kurzhals et al., Acta Pharm. Nord., 1 (5), 269-78
( 1989); US 4,797,472; EP-B 0 224 254; WO-A 87/05608; EP-B 0 170 797; EP-B 0
167
980; EP-B 0 152 872; EP-B 0 076 042; JP-A 52148032).
XS can alternatively have the following structure (V):
R2
S
\R,
R~
in which R1 to R3 have the meaning given above for formulae III and IV.
Peptide substrates of this type in which X4 = NR, O are known (cf. for example
T.
Morimoto et al., Pept. Chem., Vol. date 1989, 27'h., 387-390 ( 1999); N.
Nishino et
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_ 8 _
al., Pept. Chem., Vol. date 1988, 26'h, 21-24 ( 1989); B.J. Johnson et al., J.
Org.
Chem., 35 ( 1 ), 255-257 ( 1970) ).
In a preferred embodiment the peptide substrate is a thrombin substrate of the
general formula VI
R~
N ~ ~ NH-CO-Arg-Pro-Gly-Tos (VI)
RZ ~ R3
in which R1 denotes an alkyl residue or hydrogen,
Rz denotes a hydroxy alkyl residue or hydrogen and
R~ denotes hydrogen, halogen, alkoxy or alkylthio.
Suitable protease substrates are generally compounds which are composed of a
peptide residue that can be cleaved off by a protease of the blood coagulation
system
and are bound via an amide bond at the carboxyl end to substituted anilines
and in
particular to a phenylenediamine residue.
In addition to so-called global tests in which the clotting time is determined
as such
and which determine the activity of the protease thrombin for this purpose,
appropriate alternative substrates known to a person skilled in the art can be
used as
a basis for other coagulation tests e.g. an anti-factor Xa test or to
determine
individual coagulation factors. For this it is necessary to adapt the peptide
part of
the substrate to the protease to be determined. For the electrochemical
determination of coagulation or individual coagulation factors according to
the
invention it is important that a cleavage product of the peptide substrate
that can be
determined electrochemically is formed and detected.
The global tests include in particular the following tests: PT (prothrombin
time
test), aPTT (activated partial prothrombin time test) and ACT (activated
clotting
time test).
CA 02400651 2002-08-19
_g_
The invention is elucidated in more detail by the following examples. All
examples
are described using whole blood as the sample material. The sensors and
methods
according to the invention are, however, also suitable for citrate plasma if
calcium is
added to the formulation of the test support or to the sample. Furthermore it
is
possible according to the invention, in addition to the method mentioned in
the
examples, of applying the reagent to the electrodes (activators and substrate
are
both applied to the electrodes and subsequently dried), to not directly apply
and dry
the reagent to the electrode but in the vicinity of the electrodes for example
next to
the electrodes on a flat substrate. In this case the reagent is transported to
the
electrodes with the blood sample during the measurement. Alternatively it is
possible to incorporate the reagent in porous materials such as fleeces,
papers,
membranes and such like, and for example to incorporate the reagent in a
detachable form. In this case the blood or plasma sample has to flow through
these
materials before contact with the electrodes and in this process take up the
reagent.
It is also possible to apply individual components of the reagent to different
compartments of the test support, for example the thrombin substrate on or
directly adjacent to the electrode, but the activator spatially separated
therefrom
(e.g. further removed from the electrode) or to impregnate both substances in
different layers (e.g. membranes or fleeces).
Example 1:
Manufacture of a coagulation sensor for a PT test
I. Description of the electrochemical function
a) Open two electrode system for "top dosing" sample application. Flat
construction with 2 Pd electrodes. An Ag/AgCI paste (type: Acheson Electrodag
6037 SS) is dispensed over the whole area of a Pd electrode such that the
entire
surface of this Pd electrode is covered by Ag/AgCI.
The electrochemical operation occurs quasi-potentiostatically preferably as a
2 electrode system in which the Ag/AgCI electrode operates simultaneously as a
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reference and counterelectrode and the Pd electrode operates as a working
electrode.
A constant voltage is applied to this two electrode system and the current is
measured over time.
b) Replacement of the silver-silver chloride reference electrode by a soluble
substance that can be reduced at the counterelectrode (type 2 mediator):
A soluble reducible substance is used which accepts electrons at the counter-
electrode and thus ensures current transport through the counterelectrode. At
the same time the reduction potential that can be reached at the working
electrode is limited by the reduction potential of this substance and by the
constant voltage applied to the sensor between the working and the counter-
electrode. The p-aminoaniline to be detected at the working electrode by
oxidation must be within the achievable oxidation potential. The potential at
the working electrode against a hypothetical silver-silver chloride reference
electrode must be such that the current through the working electrode (in this
case anode) and the current through the counterelectrode (in this case
cathode)
are identical in terms of magnitude. The reducible substance should not at the
same time be oxidizable at the working electrode within the maximum
achievable oxidation potential since otherwise it would be superimposed on the
signal of the p-aminoaniline cleaved in the previous enzymatic reaction.
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II. Manufacture of a dry chemistry format for a PT test
a) Formulation of the bulk reagent:
Formulation 1: Ag/AgCI electrode configuration as described in l.La):
Concentration Substance Function of the Supplier
(in brackets allowed substance
range
according to the
invention)
0.6 % (0.1-5 %) Avicel RC 591 film former FMC corporation
(carboxylated
micro-
crystalline
cellulose)
2 % (0-5 %) Natrosol 250 thickener Aqualon
M
0.05 % (0-5 %) Polyox 750 film former Union Carbide
0.9 % (0.05-5 Triton X100 detergent Sigma
%)
200 mM HEPES buffer Roche
(10-500 mM)
0.1 % (0.01-1 BSA stabilizing proteinRoche
%)
(bovine serum
albumin)
0.1 ~g/ml rhTF activator of Stago
the
(0.01-2 ~g/ml) (human recombinantextrinsic path
of
tissue factor) plasmatic
coagulation
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Formulation 2: to be used with the type 2 mediator according to l.Lb)
Concentration Substance Function of Supplier
the
(in brackets allowed substance
range
according to the
invention)
0.6 % (0.1-5 %) Avicel RC 591 film former FMC corporation
(carboxylated
micro-
crystalline cellulose)
2 % (0-5 %) Natrosol 250 thickener Aqualon
M
0.05 % (0-5 %) Polyox 750 film former Union Carbide
0.9 % (0.05-5 Triton X100 detergent Sigma
%)
200 mM HEPES buffer Roche
(10-500 mM)
0.1 % (0.01-1 BSA stabilizing Roche
%) protein
(bovine serum
albumin)
0.1 ~g/ml rhTF activator of Stago
the
(0.01-2 pg/ml) (human recombinantextrinsic path
of
tissue factor) plasmatic
coagulation
50 mM p-nitroso-bis type 2 mediatorRoche
(1-250 mM) hydroxyethylaniline
b) Metering and drying
~l of these suspensions were metered on the sensors described under l.La)
and b) and dried on a belt dryer or in a drying cabinet at 30 to 50°C.
Formulation 1 is suitable for the sensor according to l.La); formulation 2 is
suitable for the sensor according to l.Lb).
c) When the sensor obtained in this manner is used to determine blood
coagulation a function curve (current versus time) is obtained as shown in
fig. 1. Fig. 2 shows the function curve of a sensor with a reference
electrode.
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- 1~ -
Example 2
Manufacture of a dry chemistry format for an ACT test:
a) Formulation of the bulk reagent
Formulation (alternative 1):
Concentration Substance Function of Supplier
the
(in brackets allowed substance
range
according to the
invention)
0.6 % (0.1-5 %) Avicel RC 591 film former FMC corporation
(carboxylated
micro-
crystalline cellulose)
2 % (0-5 %) Natrosol 250 thickener Aqualon
M
0.05 % (0-5 %) Polyox 750 film former Union Carbide
0.9 % (0.05-5 Triton X100 detergent Sigma
%)
200 mM HEPES buffer Roche
(10-500 mM)
0.1 % (0.01-1 BSA stabilizing Roche
%) protein
(bovine serum
albumin)
0.5 % Celite activator of Sigma
the
intrinsic path
of
plasmatic
coagulation
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Formulation (alternative 2):
Concentration Substance Function of Supplier
the
substance
30 mg/ml sucrose stabilizer Sigma
mg/g gelatin film former American Gelatin
0.1 mg/ml Triton X100 detergent Sigma
40 mg/ml glycine buffer Roche
1 % BSA stabilizing Roche
protein
(bovine serum
albumin)
0.1 pg/ml rhTF activator of Stago
the
(human recombinantextrinsic path
of
tissue factor) plasmatic
coagulation
3 mg/ml bovine sulfatideactivator of Sigma
the
intrinsic path
of
plasmatic
coagulation
b) Metering and drying
5 ~1 of one of these suspensions was metered on the sensor described under 1.
(variant la, i.e. with Ag/AgCI reference electrode) and dried on a belt dryer
or in a
drying cabinet at 30 to 50°C.
Example 3:
Amplification of the measurement signal:
A p-aminoaniline (phenylenediamine) is released from the thrombin substrate by
the action of the coagulation cascade. This is oxidized on the working
electrode and
the electrons released in this process are detected. The primary oxidation
product is
a quinone diamine. Enzymatic recycling of this oxidation product into the
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phenylenediamine by a dye-oxidoreductase such as glucose-dye-oxidoreductase
(GIucDOR) enables a renewed oxidation on the electrode and thus an
amplification
of the original signal.
a) Formulation of the bulk reagent:
Concentration Substance Function of Supplier
the
(in brackets allowed substariCe
range
according to the
invention)
0.6 % (0.1-5 %) Avicel RC 591 film former FMC corporation
(carboxylated
micro-
crystalline cellulose)
2 % (0-5 %) Natrosol 250 thickener Aqualon
M
0.05 % (0-5 %) Polyox 750 film former Union Carbide
0.9 % (0.05-5 Triton X100 detergent Sigma
%)
200 mM HEPES buffer Roche
( IO-500 mM)
0.1 % (0.01-1 BSA stabilizing Roche Diagnostics
%) protein
(bovine serum
albumin)
0.1 ~g/ml rhTF activator of Stago
the
(0.01-2~g/ml) (human recombinantextrinsic path
of
tissue factor) plasmatic
coagulation
1.2 KU/g glucose-dye- enzyme Roche
(0.01-10 KU/g) oxidoreductase
(GIucDOR)
The glucose required as a substrate for the amplification enzyme GIucDOR is a
component of the sample (ca. 10 mM) and was therefore not incorporated into
the
formulation. However, in principle it is possible to add glucose.
b) Metering and drying
~1 of this suspension was metered on the sensor described under l.La. and
dried
on a belt dryer or in a drying cabinet at 30 to 50°C.
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Example 4:
Electochemical ACT test
a) Test composition
The test composition (sensor) used in this example corresponded to the sensor
described in l.Lb) (use of a type 2 mediator).
b) Formulation of the bulk reagent
Concentration Substance Function of Supplier
the
(in brackets allowed substance
range
according to the
invention)
0.6 % (0.1-5 %) Avicel RC 591 film former FMC corporation
(carboxylated
micro-
crystalline cellulose)
2 % (0-5 %) Natrosol 250 thickener Aqualon
M
0.05 % (0-5 %) Polyox 750 film former Union Carbide
0.9 % (0.05-5 Triton X100 detergent Sigma
%)
200 mM HEPES buffer Roche
( 10-500 mM)
0.1 % BSA stabilizing Roche
protein
(0.01-1 %) (bovine serum
albumin)
0.2 pg/ml rhTF activator of Stago
the
(0.01-2 ~tg/ml) (human recombinantextrinsic path
of
tissue factor) plasmatic
coagulation
6 mg/ml bovine sulfatideactivator of Life Science
the
(0.5 - 50 mg/ml) extrinsic path Research
of
plasmatic
coagulation
50 mM p-nitroso-bis- type 2 mediatorRoche
( 1- 250 mM) hydroxyethylaniline
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- 17 -
c) Metering and drying
~1 of this suspension was metered on the sensor described under a) and dried
on a
belt dryer or in a drying cabinet at 30 to 50°C.
d) Results of the measurement for the ACT test
An ACT test was carried out with the sensor described above using whole blood
samples to which 1 to 6 U heparin per ml was added (heparin spike). A
potential of
300 mV was applied and the time was read at a threshold value of 0.5 ~A. The
results are shown in the following table:
Heparin concentration Clotting time (s]
[U/ml]
1 47
2 100
3 172
4 235
5 279
6 353
CA 02400651 2002-08-19
Exam 1p a 5:
Electochemical aPTT test
a) Test composition:
The test composition used in this example corresponded to the composition
described in l.Lb).
b) Formulation of the bulk reagent:
Concentration Substance Function of the Supplier
(in brackets allowed substance
range
according to the
invention)
0.6 % (0.1-5 %) Avicel RC 591 film former FMC corporation
(carboxylated
micro-
crystalline
cellulose)
2 % (0-5 %) Natrosol 250 thickener Aqualon
M
0.05 % (0-5 %) Polyox 750 film former Union Carbide
0.9 % (0.05-5 Triton X100 detergent Sigma
%)
200 mM HEPES buffer Roche
( 10-500 mM)
0.1 % BSA stabilizing proteinRoche Diagnostics
(0.01-I %) (bovine serum
albumin)
0.6 mg/ml soy bean activator of Sigma
the
(0.05-10 mg/ml) phosphatides extrinsic path
of
plasmatic
coagulation
1 mg/ml bovine sulfatideactivator of Life Science
the
(0.1-10 mg/ml) extrinsic path Research
of
plasmatic
coagulation
50 mM p-nitroso-bis- type 2 mediator Roche
( 1- 250 mM) hydroxyethylaniline
CA 02400651 2002-08-19
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c) Metering and drying
~l of this suspension was metered on the sensor described under l.Lb) and
dried
on a belt dryer or in a drying cabinet at 30 to 50°C.
d) Results of the measurement for the aPTT test
An aPTT test was carried out with the sensor described above using whole blood
samples to which 0 to 0.35 U heparin per ml was added (heparin spike). A
potential
of 300 mV was applied and the time was read at a threshold value of 0.5 ~A.
The
results are shown in the following table:
Heparin concentration Clotting time
[U/ml] [s]
0 59
0.15 79
0.25 117
0.35 164
Example 6:
Compensation of the haematocrit effect
I. Description of the method:
In chronoamperometric or voltammetric measuring methods the current resulting
from an oxidation or reduction depends on the haematocrit when using whole
blood as the analyte. Hence in evaluation methods which are based on a defined
CA 02400651 2002-08-19
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current threshold to determine the clotting time, different clotting times are
determined depending on the haematocrit.
The haematocrit can be measured by a special measurement algorithm and used to
correct the current measurements. For this purpose a brief measurement of the
apparent resistance of the sensor wetted with blood is carried out before the
actual
chronoamperometric measurement. For this an alternating voltage with a
frequency
of 2 kHz and an amplitude of about 10 mV is applied between the working
electrode and the counterelectrode and the resulting effective value of the
alternating current flowing through the sensor is measured. The apparent
resistance
Z of the sensor filled with the blood sample is obtained by dividing the
effective
value of the applied voltage with the effective value of the alternating
current. This
apparent resistance Z is dependent on the temperature and the haematocrit of
the
blood. Under thermostatted isothermal conditions it is thus possible to
measure the
haematocrit in the blood independent of the concentration of the actual
electron
carrier (mediator).
The current values of the actual chronoamperometric detection measurement
(imess)
can then be corrected using the following formula in order to thus obtain
results
(i~orl) which are independent of the haematocrit content:
lcorr = lmess X ( (Z - Zmedian) x ~.~~ 1 -t- 1 )
in which Z is the apparent resistance of the actual measurement and Zmeaian is
the
apparent resistance of the average of many blood samples and is derived as the
result of a batch calibration.
II. Experimental procedure
IL 1 Preparation of the dry chemistry format for the PT test:
Compensation for the haematocrit effect is demonstrated using a PT test as an
example. The test composition used in this example corresponded to the
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composition described in example 1 under Ib). The formulation used was
identical
to formulation 2 described in section IIa) of example I.
IL2. Setting the haematocrit values
Fresh venous blood was cooled on an ice bath to 0°C. Aliquots were
separated in a
bench centrifuge into erythrocytes and cell pellet. High haematocrits were set
by
removing part of the plasma and resuspending the cells in the remaining
plasma.
Low haematocrits were obtained by adding plasma which had been obtained from
another aliquot of the same blood as already described.
The experimental results are shown in the following table:
Haematocrit (%] Clotting times [s]
uncorrected corrected
35 39 52
45 45 53
65 78 56
Example 7:
Voltammetric method
In addition to the described amperometric measuring methods, it is also
possible to
use voltammetric measuring methods. In this case the voltage between the
electrodes is not set to be constant, but rather the voltage is changed
linearly from
an initial value to a final value and subsequently returned to the initial
value. This
process can be repeated several times over the entire measurement period. In
this
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method the current is then plotted against the voltage and when this is
repeated,
one obtains a nested set of current-voltage curves (cyclovoltammograms). If
the
voltage range is suitable, oxidation peaks and reduction peaks of the electron
carrier
are formed in these curves.
An example of such consecutive voltammograms is shown in fig. 2. A sweep
through the potential range 100 to 600 mV and back was repeated over the
entire
measurement period.
The height of these peaks is directly proportional to the concentration of the
electron carrier provided that other redox-active substances are not co-
oxidized or
co-reduced in the covered potential range and thus additionally contribute to
the
current. Such an interference may be ignored when measuring a concentration
change.
If the current values for the peak maxima or the areas enclosed by the curves
(corresponding to the charge turnover) of the individual current voltage loops
are
plotted against time, one also obtains a picture of the concentration change
of the
electron carrier over the measurement period in a time raster given by the
cycle
period. The charge content (sum of the current values/measurement density) of
the
consecutive cyclic voltammograms is plotted over time in fig.3. The difference
in
the time course between the normal coagulation range and the result of a
Marcumar
test person (with retarded blood clotting) can be seen.
The time at which the charge of a cyclic voltammogram starts to increase and
how
rapidly this occurs is of primary interest in order to determine or read a
clotting
time. Hence this means when does coagulation start (thrombin is activated) and
how rapidly does this occur (how rapidly is how much thrombin additionally
activated). For this reason, as shown in fig. 4, the time derivative is
calculated from
the curves of fig. 3 i.e. dQ to dt. The maximum slope is the maximum in the
time
derivative. The time t of the maximum can be read as the clotting time.
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The following result is then obtained for the examples shown in figures 3 and
4:
Time until maximum
dQ/dt
Measurement Normal range Marcumar test person
1 74 208
2 70 200
3 72 196
4 74 200
72 196
6 74 188
7 74 208
8 74 216
9 74 204
74 234
11 76 218
