U.S. patent application number 14/153739 was filed with the patent office on 2014-05-08 for blood coagulation time prolonging agent.
This patent application is currently assigned to SEKISUI MEDICAL CO., LTD.. The applicant listed for this patent is SEKISUI MEDICAL CO., LTD.. Invention is credited to Chizuru MORIKAWA, Remi NAKAMURA, Mitsuaki YAMAMOTO.
Application Number | 20140127727 14/153739 |
Document ID | / |
Family ID | 43126036 |
Filed Date | 2014-05-08 |
United States Patent
Application |
20140127727 |
Kind Code |
A1 |
MORIKAWA; Chizuru ; et
al. |
May 8, 2014 |
BLOOD COAGULATION TIME PROLONGING AGENT
Abstract
Provided is a reagent, which prolongs a blood coagulation time
sufficiently and enhances an optical change, thereby enabling a
correct and high-sensitive blood coagulability test. The present
invention provides a blood coagulation time prolonging agent,
including, as an active ingredient, a guanidine compound
represented by the following formula (1) or an acid addition salt
thereof (in the formula, R.sup.1 represents a hydrogen atom, an
amino group, or an alkyl group which may have a substituent).
Inventors: |
MORIKAWA; Chizuru;
(Ryugasaki-shi, JP) ; NAKAMURA; Remi;
(Ryugasaki-shi, JP) ; YAMAMOTO; Mitsuaki;
(Ryugasaki-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SEKISUI MEDICAL CO., LTD. |
Chuo-ku |
|
JP |
|
|
Assignee: |
SEKISUI MEDICAL CO., LTD.
Chuo-ku
JP
|
Family ID: |
43126036 |
Appl. No.: |
14/153739 |
Filed: |
January 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13321732 |
Nov 21, 2011 |
|
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PCT/JP10/03401 |
May 20, 2010 |
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14153739 |
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Current U.S.
Class: |
435/13 |
Current CPC
Class: |
C07C 281/16 20130101;
G01N 33/86 20130101; C12Q 1/56 20130101; A61K 31/155 20130101; C07C
279/02 20130101; A61P 7/02 20180101; C07C 279/14 20130101 |
Class at
Publication: |
435/13 |
International
Class: |
G01N 33/86 20060101
G01N033/86 |
Foreign Application Data
Date |
Code |
Application Number |
May 20, 2009 |
JP |
2009-121834 |
Claims
1-21. (canceled)
22. A method of measuring blood coagulation, comprising: (a) adding
to a plasma specimen a guanidine compound represented by formula
(1) or an acid addition salt thereof: ##STR00004## wherein R.sup.1
represents a hydrogen atom, an amino group, or an alkyl group which
may optionally have a substituent; (b) adding to the plasma
specimen a blood coagulation activator, wherein the blood
coagulation activator induces the formation of a network of fibrin
in the plasma specimen, wherein the network of fibrin in the plasma
specimen increases turbidity in the plasma specimen, and wherein
the guanidine compound represented by formula (1) or an acid
addition salt thereof enhances turbidity in the plasma specimen and
prolongs the blood coagulation time; and (c) measuring a change,
.DELTA.H, in the scattered light intensity from the plasma specimen
to determine the coagulation time of the plasma specimen.
23. The method of claim 22, wherein R.sup.1 represents a hydrogen
atom, an amino group, an alkyl group, an alkyl group substituted
with a carboxy group and an amino group, or an alkyl group
substituted with an alkoxycarbonyl group and an amino group.
24. The method of claim 22, further comprising adding a specimen
diluent to the plasma specimen when adding the guanidine compound
represented by formula (1) or an acid addition salt thereof:
25. The method of claim 22, wherein the blood coagulation activator
comprises thrombin.
26. The method of claim 22, wherein the guanidine compound
represented by formula (1) or an acid addition salt thereof is
guanidine or an acid addition salt thereof.
27. The method of claim 22, wherein guanidine compound represented
by formula (1) or an acid addition salt thereof is guanidine
hydrochloride.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a reagent for measuring
blood coagulability typified by a reagent for measuring fibrinogen;
specifically relates to a blood coagulation time prolonging agent
used in a blood coagulation activator-containing reagent and/or in
a specimen diluent for measuring blood coagulability, and to a
reagent for measuring blood coagulability using this blood
coagulation time prolonging agent.
BACKGROUND OF THE INVENTION
[0002] A blood coagulation mechanism is broadly divided into two
pathways in general. One is an intrinsic pathway, which is
initiated by contact activation of blood coagulation factor XII
with a foreign substance, and finally produces thrombin through
multistep reactions, and the other is an extrinsic pathway, which
is initiated by activation of blood coagulation factor X with blood
coagulation factor VII and tissue thromboplastin, and produces
thrombin in the same manner as described above (FIG. 1). In both of
the pathways, coagulation eventually occurs by conversion of
fibrinogen to fibrin through an action of produced thrombin. In
order to clarify the presence or absence of abnormality or a cause
of the abnormality in such blood coagulation mechanism, some blood
coagulability tests using a blood coagulation activator are
available and widely used in actual clinical examinations.
[0003] The blood coagulation activator and the blood coagulability
tests using the same are as follows:
1) blood coagulability tests using thrombin
[0004] a fibrinogen measurement, an ATIII measurement, and a
thrombin time measurement;
2) blood coagulability tests using tissue thromboplastin
[0005] a prothrombin time measurement, an activity measurement of
factor II, V, VII, or X using the prothrombin time measurement, and
a measurement of complex factors (a thrombo test, a hepaplastin
test, and the like); and
3) blood coagulability tests using phospholipids
[0006] a partial thromboplastin time measurement, an activated
partial thromboplastin time measurement, an activity measurement of
factor VIII, IX, XI, or XII, prekallikrein, or high molecular
kininogen using the activated partial thromboplastin time
measurement, a viper venom time measurement, a quantitative
determination of factor X using the viper venom time measurement, a
lupus anti-coagulant (LA) measurement using a diluted viper venom
time measurement, a protein C activity measurement, and a
thromboplastin generation test.
[0007] In any of the above-mentioned tests 1) to 3), a time from
initiation of coagulation by mixing a reagent including a blood
coagulation activator and the like with a specimen from a patient
to final conversion of fibrinogen into fibrin to be deposited is
measured.
[0008] Methods of detecting coagulation in the blood coagulability
test can be broadly divided into a mechanical detection method and
an optical detection method. The mechanical detection method refers
to a method including: monitoring by a magnetic force or the like a
magnetic substance or the like loaded into a reaction solution; and
detecting a decrease in mobility of the magnetic substance due to
an increase in viscosity by coagulation. The optical detection
method refers to a method including detecting white turbidity of a
reaction solution due to coagulation as a change in transmitted
light or scattered light, and is most widely used because the
method is relatively simple. These are generally detected by an
automated analyzer at present. One example of a change curve for
scattered light intensity obtained in a scattered light detection
method is shown in FIG. 2. In the figure, the point A indicates a
point at which the coagulation is initiated by mixing a reagent
including a blood coagulation activator, subsequently fibrin
deposition is initiated through multistep reactions, and as a
result, a change in scattered light intensity appears after the
point B. Fibrinogen is in turn consumed and almost depleted in the
reaction solution.
[0009] Then, the scattered light intensity exhibits no change, the
curve becomes flat as shown at the point C, and the coagulation is
terminated. A coagulation time is calculated with known calculation
parameters based on such change curve for the scattered light
intensity (Patent document 1). Here, it is obvious that when a
maximum of the change in scattered light intensity is defined as
.DELTA.H, as the .DELTA.H is larger, the coagulation is detected
more sharply and more correct detection becomes possible. Thus, the
reagent for measuring blood coagulability desirably has a property
in which an optical change is largely displayed. However, when the
change from the point A to the point C occurs in an extremely short
period of time, it is impossible to measure a coagulation time with
good accuracy. Thus, a substance for prolonging the coagulation
time is generally added to adjust the coagulation time to a desired
time. A blood coagulation time prolonging agent is exemplified by
halide salts of alkali metals and alkali earth metals, including
sodium chloride in Patent document 2, and by sodium propionate in
Patent document 3. However, there has been a problem in that such
conventional blood coagulation time prolonging agent also has a
large negative effect of reducing .DELTA.H in an addition
dose-dependent manner.
[0010] For the purpose of solving the problems in those
conventional methods, there is proposed a method including using a
reagent supplemented with a high molecular material such as
polyethylene glycol, polyvinyl alcohol or high molecular
polysaccharide (Patent documents 4 and 5).
PRIOR ART
Patent Document
[0011] Patent Document 1: JP-08-15263 A [0012] Patent Document 2:
JP2994557 B2 [0013] Patent Document 3: JP3330685 B2 [0014] Patent
Document 4: JP3074611 B2 [0015] Patent Document 5: JP05-60763 A
SUMMARY OF INVENTION
[0016] However, there are problems in that, in the method including
adding a high molecular material to a reagent, the viscosity of the
reagent itself is increased to reduce the accuracy of sampling the
reagent with a pipette or a reagent probe, and in that a difference
in turbidity between non-coagulated and coagulated solutions is not
obtained sufficiently and consequently an optical change is not
sufficiently enhanced.
[0017] Therefore, it is an object of the present invention to
provide a reagent having a composition that prolongs a blood
coagulation time to a desired time and enhances an optical change.
This can contribute to enhancement of correctness in a blood
coagulability test.
[0018] Thus, the inventors of the present invention have searched a
blood coagulation time prolonging agent which enhances an optical
change using various compounds, and have found that a guanidine
compound having a specific structure or a salt thereof has not only
an action of prolonging a blood coagulation time but also an action
of enhancing an optical change upon coagulation, and the use
thereof as an additive for a reagent for measuring blood
coagulability allows the blood coagulability to be measured
correctly with high sensitivity, and have completed the present
invention.
[0019] That is, the present invention provides a blood coagulation
time prolonging agent, including, as an active ingredient, a
guanidine compound represented by the following formula (1) or an
acid addition salt thereof:
##STR00001##
[0020] (in the formula, R.sup.1 represents a hydrogen atom, an
amino group, or an alkyl group which may have a substituent).
[0021] Further, the present invention provides a blood coagulation
activator-containing reagent or a specimen diluent for measuring
blood coagulability, including the above-mentioned blood
coagulation time prolonging agent.
[0022] In addition, the present invention provides a blood
coagulation activator-containing reagent for measuring fibrinogen
including thrombin as the blood coagulation activator or a specimen
diluent for measuring fibrinogen, containing the above-mentioned
blood coagulation time prolonging agent.
[0023] Further, in one aspect, the present invention provides a use
of a guanidine compound represented by the following formula (1) or
an acid addition salt thereof for prolonging a blood coagulation
time:
##STR00002##
(in the formula, R.sup.1 represents a hydrogen atom, an amino
group, or an alkyl group which may have a substituent).
[0024] In one embodiment, the above-mentioned guanidine compound or
acid addition salt thereof is used in combination with the blood
coagulation activator. In another embodiment, the above-mentioned
guanidine compound or acid addition salt thereof is used in
combination with the specimen diluent for measuring blood
coagulability.
[0025] Further, in one aspect, the present invention provides a
method of prolonging a blood coagulation time, including adding to
a plasma specimen a guanidine compound represented by the following
formula (1) or an acid addition salt thereof:
##STR00003##
(in the formula, R.sup.1 represents a hydrogen atom, an amino
group, or an alkyl group which may have a substituent).
[0026] In one embodiment, the addition of the above-mentioned
guanidine compound or acid addition salt thereof is carried out in
combination with the specimen diluent for measuring blood
coagulability. In another embodiment, the addition of the
above-mentioned guanidine compound or acid addition salt thereof is
carried out in combination with the blood coagulation
activator.
[0027] Further, in one aspect, the present invention provides a
method of measuring blood coagulability, including the steps of:
preparing a reaction solution containing a plasma specimen, a blood
coagulation activator, and the above-mentioned guanidine compound
or acid addition salt thereof; and measuring a coagulation time of
the reaction solution.
[0028] Further, in one aspect, the present invention provides a
method of determining a fibrinogen concentration in the plasma
specimen by comparing a measured value of the coagulation time
using the plasma specimen with a measured value obtained by
similarly measuring the coagulation time using a diluted standard
solution in place of the plasma specimen.
Advantageous Effect of the Invention
[0029] When the blood coagulation time prolonging agent of the
present invention is used, fibrinogen level, PT, and APTT for blood
coagulability can be measured correlatively because the optical
change is enhanced.
BRIEF DESCRIPTION OF DRAWINGS
[0030] FIG. 1 is a view schematically showing a blood coagulation
mechanism in a clinical examination.
[0031] FIG. 2 is a graph showing one example of a change curve for
scattered light intensity obtained by a scattered light detection
method when blood coagulability is measured.
[0032] FIG. 3 is a graph showing a relationship between an addition
amount and a coagulation time of each compound.
[0033] FIG. 4 is a graph showing a relationship between a
coagulation time and a maximum of a change in scattered light
intensity when the coagulation time is controlled by the addition
of each compound.
[0034] FIG. 5 is a graph showing change curves for scattered light
intensity obtained by using reagents according to conventional
methods and a reagent of the present invention.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0035] A blood coagulation time prolonging agent of the present
invention contains a compound represented by the formula (1) or a
salt thereof as an active ingredient. In the formula (1), R.sup.1
represents a hydrogen atom, an amino group, or an alkyl group which
may have a substituent. Here, the alkyl group is preferably an
alkyl group having 1 to 6 carbon atoms, particularly preferably an
alkyl group having 2 to 5 carbon atoms. A group which may serve as
a substituent of the alkyl group is exemplified by a carboxy group,
an alkoxycarbonyl group, and an amino group. The alkyl group is
preferably substituted with both of the carboxy group and the amino
group or with both of the alkoxycarbonyl group and the amino
group.
[0036] Specific examples of the compound represented by the formula
(1) include guanidine, aminoguanidine, arginine, and an arginine
alkyl ester. Of those, aminoguanidine is particularly preferred
because an action of enhancing an optical change is large and the
correct measurement can be performed. Further, examples of the acid
addition salt of the compound of the formula (1) include a
hydrochloride, a sulfate, a nitrate, a phosphate, and a
sulfamate.
[0037] The blood coagulation time prolonging agent of the present
invention has not only an action of prolonging a blood coagulation
time but also an action of enhancing a turbidity change of a
reaction solution in the measurement of blood coagulability. This
indicates that a network of fibrin, which is a final product of
blood coagulation, is more firmly formed. Therefore, the blood
coagulation time prolonging agent of the present invention can be
used in, for example, an optical detection method including
directly detecting a turbidity change and a mechanical detection
method including monitoring a formation state of a network of
fibrin with a magnetic substance or the like. Of those, it is
preferred to use the blood coagulation time prolonging agent in the
optical detection method. Here, a method of detecting a scattered
light amount and a method of detecting a transmitted light amount
are available as the optical detection method. Of those, it is
preferred to use the blood coagulation time prolonging agent in the
method of detecting a scattered light amount.
[0038] A blood coagulability test to which the blood coagulation
time prolonging agent of the present invention can be applied may
be any test including measuring a time from the initiation of
coagulation by mixing a reagent containing a blood coagulation
activator and the like with a patient specimen to the final
conversion of fibrinogen into fibrin to be deposited. Examples
thereof include a fibrinogen measurement, an ATIII measurement, a
thrombin time measurement, a prothrombin time measurement, an
activity measurement of factor II, V, VII, or X using a prothrombin
time, a measurement of complex factors (a thrombo test, a
hepaplastin test, and the like), a partial thromboplastin time
measurement, an activated partial thromboplastin time measurement,
an activity measurement of factor VIII, IX, XI, or XII,
prekallikrein, or high molecular kininogen using the activated
partial thromboplastin time measurement, a viper venom time
measurement, a quantitative determination of factor X using the
viper venom time measurement, a lupus anti-coagulant (LA)
measurement using a diluted viper venom time measurement, a protein
C activity measurement, a thromboplastin generation test, and any
other detection of abnormal coagulation.
[0039] In the measurement in the above-mentioned blood
coagulability test, a time from the addition of a blood coagulation
activator-containing reagent (e.g., a thrombin-containing reagent
in the fibrinogen measurement) to a specimen to the deposition of
fibrin is measured. The specimen is appropriately diluted with a
specimen diluent if necessary. The blood coagulation time
prolonging agent of the present invention has only to be contained
in a measurement system. The blood coagulation time prolonging
agent may be contained in the blood coagulation
activator-containing reagent, or may be contained in the specimen
diluent. That is, for example, in the fibrinogen measurement, the
blood coagulation time prolonging agent of the present invention
may be contained in the thrombin-containing reagent, or may be
contained in the specimen diluent.
[0040] The above-mentioned blood coagulation activator-containing
reagent has only to be one used for the above-mentioned blood
coagulability test, and examples thereof include a
thrombin-containing reagent, a tissue thromboplastin-containing
reagent, and a phospholipid-containing reagent. Further, the
above-mentioned specimen diluent has only to be one used for the
above-mentioned blood coagulability test, and examples thereof
include Good's buffers such as MES, Bis-Tris, ADA, PIPES, ACES,
MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS,
Tricine, Bicine, TAPS, and CHES, a citrate buffer, a phosphate
buffer, an acetate buffer, an imidazole buffer, a barbital buffer,
saline, and water.
[0041] The blood coagulation time prolonging agent of the present
invention is preferably contained in the reaction solution so that
a coagulation time which is measurable with high accuracy is
obtained in the blood coagulability test. For example, in the
fibrinogen measurement, the blood coagulation time prolonging agent
is preferably contained in the reaction solution so that the
coagulation time is 5 to 50 seconds, preferably 7 to 20 seconds,
more preferably 9 to 15 seconds when normal plasma (concentration:
about 300 mg/dL) is measured. When aminoguanidine is used as the
blood coagulation time prolonging agent, the concentration of
aminoguanidine in the reaction solution for the fibrinogen
measurement, i.e., the concentration of aminoguanidine in a mixed
solution of a specimen, a specimen diluent, and a
thrombin-containing reagent is 10 to 500 mM, preferably 20 to 200
mM, more preferably 30 to 120 mM. When aminoguanidine is contained
in the specimen diluent, the concentration of aminoguanidine in the
specimen diluent is 10 to 900 mM, preferably 30 to 400 mM, more
preferably 50 to 200 mM.
[0042] In the prothrombin time measurement, the blood coagulation
time prolonging agent is preferably contained in the reaction
solution so that the coagulation time is 9 to 60 seconds,
preferably 9 to 30 seconds, more preferably 10 to 16 seconds when
normal plasma (activity: about 100%) is measured. In the activated
partial thromboplastin time measurement, the blood coagulation time
prolonging agent is preferably contained in the reaction solution
so that the coagulation time is 15 to 80 seconds, preferably 15 to
60 seconds, more preferably 20 to 50 seconds when normal plasma is
measured. Also in the other blood coagulability tests, the blood
coagulation time prolonging agent has to be contained in the
reaction solution so that a coagulation time suitable for a
measurement method is obtained.
[0043] In addition to the blood coagulation time prolonging agent
of the present invention, known high molecular polysaccharides and
synthetic polymers can be contained in the reagent for measuring
blood coagulability including the specimen diluent and the blood
coagulation activator-containing reagent used for the blood
coagulability test.
[0044] Specific examples of the high molecular polysaccharides
include dextran such as dextran 40, dextran 70, dextran 200000, or
dextran 500000, and Ficoll, and the like. One kind of those high
molecular polysaccharides may be used alone, or two or more kinds
thereof may be used in combination. Those high molecular
polysaccharides are each added in an amount so that the
concentration in the reagent for measuring blood coagulability is
suitably 0.01 to 10 W/V %, more preferably 0.1 to 5 W/V %.
[0045] Specific examples of the synthetic polymers include:
polyvinyl alcohol such as polyvinyl alcohol 500, polyvinyl alcohol
1500, or polyvinyl alcohol 2000; polyethylene glycol such as
polyethylene glycol 1500, polyethylene glycol 2000, polyethylene
glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, or
polyethylene glycol 20000; and polyvinylpyrrolidone. One kind of
those synthetic polymers may be used alone, or two or more kinds
thereof may be used in combination. The amount of each of those
synthetic polymers to be added is not particularly limited, and the
concentration thereof in the reagent for measuring blood
coagulability is preferably 0.01 to 10 W/V %, more preferably 0.1
to 5 W/V %.
[0046] The reagent for measuring blood coagulability can also
contain a buffer, a calcium ion, an antagonist of an anticoagulant,
and the like in addition to the blood coagulation time prolonging
agent of the present invention.
[0047] As a specific example of the buffer, a buffer having a
buffering ability in the range of pH 4 to 9 is appropriately
selected and used. For example, the buffer can be selected from
Good's buffers such as MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES,
MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine,
Bicine, TAPS, and CHES, citric acid, phosphoric acid, acetic acid,
imidazole, barbital, GTA, and the like, and one kind thereof may be
used alone, or two or more or kinds thereof may be used in
combination. The amount of the buffer to be added is not
particularly limited as long as the buffer has a buffering ability,
and the concentration in the reagent for measuring blood
coagulability is preferably 1 to 1,000 mM, more preferably 5 to 500
mM.
[0048] Water-soluble calcium compounds such as calcium chloride,
calcium lactate, calcium gluconate, calcium glucuronate, and
calcium tartrate are used as the calcium ion. One kind of those
calcium compounds may be used alone, or two or more kinds thereof
may be used in combination. The amount of each of the calcium
compounds to be used is not particularly limited as long as the
calcium compounds can help a coagulation reaction, and the
concentration thereof in the reagent for measuring blood
coagulability is preferably 5 mM to 100 mM, and more preferably 10
mM to 50 mM.
[0049] Protamine, polybrene (hexadimethrine bromide), and the like
are used as the antagonist of the anticoagulant. One kind of those
antagonists of the anticoagulant may be used alone, or two or more
kinds thereof may be used in combination. The amount of the
antagonist of the anticoagulant to be used is not particularly
limited as long as the antagonist can sufficiently neutralize an
anticoagulant such as heparin contained in specimen plasma, and the
concentration in the reagent for measuring blood coagulability is
preferably 10.sup.-5 to 10.sup.-2 W/V %, more preferably
5.times.10.sup.-4 to 5.times.10.sup.-3 W/V %.
[0050] Further, the reagent for measuring blood coagulability may
be a liquid product, a frozen product, or a dried product, and the
dried product is dissolved by adding purified water or a
buffer.
[0051] Further, an appropriate preservative may be added to the
reagent for measuring blood coagulability. The preservative can be
selected from ciprofloxacin, propionic acid, sodium benzoate,
sodium azide, ProClin 300, and the like, and one kind thereof may
be used alone, or two or more kinds thereof may be used in
combination. Further, salts such as sodium chloride, common
stabilizers such as amino acids and sugars, and the like may be
contained as needed.
[0052] The concentration described above is the concentration in
the solution, and the concentration of the dried product or the
like is the concentration when dissolved in water, a buffer, or the
like before use.
[0053] The measurement of blood coagulability of the present
invention can be conducted with a usual method. Taking the
fibrinogen measurement as an example, a standard solution is
diluted with a specimen diluent 5-fold, 10-fold, and 20-fold (a
dilution rate can be adjusted appropriately). Next, two volumes of
each diluted standard solution is warmed at 37.degree. C. for 3
minutes, one volume of a thrombin reagent previously warmed at
37.degree. C. is added, and a coagulation time is measured.
Subsequently, a measured value of the each diluted standard
solution is plotted on a graph. A plasma specimen is diluted with a
specimen diluent 10-fold (a dilution rate can be adjusted
appropriately), the diluted specimen is measured likewise, and the
concentration can be obtained from the graph based on the obtained
coagulation time.
EXAMPLES
[0054] Hereinafter, the present invention is described in more
detail by way of examples, but the present invention is not limited
to these examples.
Example 1
(1) Identification of Coagulation Time Prolonging Action
[0055] When a fibrinogen concentration in a plasma specimen is
measured by a thrombin time method, in general, thrombin is added
to plasma to measure a coagulation time, and the concentration is
determined from a standard curve prepared using standard solutions
having known fibrinogen concentrations. In the procedure, 90 .mu.L
of a specimen diluent are added to and mixed with 10 .mu.L of
specimen plasma, subsequently 50 .mu.L of a thrombin-containing
reagent are added as a blood coagulation activator-containing
reagent, and a coagulation time is measured.
[0056] In this example, commercially available normal plasma
(Coagtrol N, manufactured by Sysmex Corporation) as the plasma
specimen, a Coagpia Fbg thrombin reagent (manufactured by Sekisui
Medical Co., Ltd.) as the thrombin-containing reagent, and a
full-automated blood coagulation time analyzer Coagrex 800
(manufactured by Sysmex Corporation) as a coagulation time
measurement apparatus were used. This apparatus employs a scattered
light detection method, and change data of scattered light
intensity with time can also be obtained along with the coagulation
time.
[0057] When an HEPES buffer (pH 7.5) was used as the specimen
diluent, the commercially available normal plasma coagulated in
several seconds (4.6 seconds). When aminoguanidine hydrochloride as
the blood coagulation time prolonging agent was added to the HEPES
buffer (pH 7.5), the coagulation time was prolonged in an addition
concentration-dependent manner in the same manner as in
conventional methods using sodium chloride and sodium bromide
(results are shown in FIG. 3). That is, it is found that the
present invention has the same coagulation time prolonging action
as the conventional methods.
(2) Identification of Change in Light Intensity
[0058] A maximum of a change in scattered light intensity was
calculated as .DELTA.H, and its relationship with the coagulation
time was shown in FIG. 4. .DELTA.H was remarkably decreased along
with the prolonged coagulation time in the conventional methods
using sodium chloride and sodium bromide, while large .DELTA.H was
kept in the present invention. In other words, when the changes are
compared in the similar coagulation time, the change in scattered
light intensity is larger in the present invention than in the
conventional methods, and it is found that the coagulation time can
be measured more correctly in the present invention.
Example 2
[0059] Commercially available normal plasma was measured in the
same manner as in Example 1 using the specimen diluent (HEPES
buffer, pH 7.5) in which the amount of the blood coagulation time
prolonging agent added was adjusted so as to obtain the similar
coagulation time. The comparative results of the maximum of a
change .DELTA.H in scattered light intensity and of the
simultaneous reproducibility of the measurements between in the
conventional method and in the present invention were shown in
Table 1. As compared with the conventional methods, it is found
that the blood coagulation time prolonging agent of the present
invention, i.e., arginine hydrochloride, arginine methyl ester,
guanidine hydrochloride, guanidine phosphate, guanidine sulfamate,
or aminoguanidine hydrochloride drastically enhances .DELTA.H and
further enhances the accuracy of the measurement (simultaneous
reproducibility or the like). It was also found that aminoguanidine
hydrochloride had a particularly excellent effect of improving the
accuracy of the measurement (simultaneous reproducibility or the
like).
TABLE-US-00001 TABLE 1 Variation coefficient of Coagulation
measured .DELTA.H time values Conventional Sodium chloride: 2,348
10.7 4.47% method (1) 145 mM Conventional Sodium bromide: 2,174
11.3 * method (2) 110 mM Conventional Potassium bromide: 2,127 11.4
* method (3) 105 mM Conventional Potassium chloride: 2,170 10.8 *
method (4) 135 mM Conventional Lithium chloride: 2,305 10.8 *
method (5) 135 mM Method of the Guanidine 4,575 10.3 3.39% present
hydrochloride: invention (1) 90 mM Method of the Guanidine 4,217
10.4 2.36% present phosphate: invention (2) 30 mM Method of the
Guanidine 5,167 11.2 3.29% present sulfamate: invention (3) 55 mM
Method of the Aminoguanidine 3,480 9.7 1.94% present hydrochloride:
invention (4) 90 mM Method of the Arginine 3,529 10.3 2.77% present
hydrochloride: invention (5) 55 mM Method of the Arginine methyl
6,130 11.0 3.22% present ester: invention (6) 17 mM Single
measurement was used in conventional methods (2) to (5) and ten
times measurement was used in the others. * Cannot be
calculated
Example 3
[0060] A specimen diluent containing sodium chloride as the blood
coagulation time prolonging agent was defined as a conventional
method A and a specimen diluent further containing polyethylene
glycol (molecular weight: 20000) (hereinafter, referred to as PEG
20000) as an enhancer of a change in light intensity in the
conventional method A was defined as a conventional method B, and
the specimen diluents were compared with a specimen diluent
employing aminoguanidine hydrochloride (present invention) which
served as both of the blood coagulation time prolonging agent and
the enhancer of a change in light intensity. It should be noted
that an HEPES buffer (pH 7.5) was used as a buffer in all of the
cases. Further, the concentration of PEG 20000 to be added in the
conventional method B was set to 0.1%, because a large amount of
PEG 20000 could increase the viscosity of diluent and reduce the
accuracy of sampling. The coagulation time of commercially
available normal plasma was measured in the same manner as in
Example 1 using each specimen diluent. As a result, a change curve
for scattered light intensity as shown in FIG. 5 was obtained.
Further, the coagulation time and .DELTA.H were shown in Table 2.
The change of the scattered light intensity in the conventional
method B with the addition of PEG 20000 became larger than in the
conventional method A without the addition. However, it is found
that, by employing the present invention, the change of the
scattered light intensity is more drastically enhanced and the
coagulation time can be measured more correctly.
TABLE-US-00002 TABLE 2 Conventional Conventional The present method
A method B invention Coagulation time NaCl 150 mM NaCl 150 mM
Aminoguanidine prolonging agent hydrochloride Enhancer of change --
PEG 90 mM in light intensity 20000 0.1% Coagulation time 11.6
seconds 10.4 seconds 10.4 seconds Change in light 2,194 2,244 3,444
intensity .DELTA.H Mean of three times measurement
* * * * *