U.S. patent application number 15/219846 was filed with the patent office on 2018-12-06 for method for measuring clotting time, measurement device for clotting time, and reagent kit.
This patent application is currently assigned to SYSMEX CORPORATION. The applicant listed for this patent is SCHOOL JURIDICAL PERSON HIGASHI-NIPPON-GAKUEN, SYSMEX CORPORATION. Invention is credited to Masahiro IEKO, Osamu KUMANO, Takeshi SUZUKI, Haruki YAMAGUCHI.
Application Number | 20180348237 15/219846 |
Document ID | / |
Family ID | 56557557 |
Filed Date | 2018-12-06 |
United States Patent
Application |
20180348237 |
Kind Code |
A9 |
KUMANO; Osamu ; et
al. |
December 6, 2018 |
METHOD FOR MEASURING CLOTTING TIME, MEASUREMENT DEVICE FOR CLOTTING
TIME, AND REAGENT KIT
Abstract
Disclosed is a method for measuring a clotting time, including
the steps of: (A) mixing a blood sample, an activator, a
phospholipid, and a nickel ion-forming compound to obtain a
specimen; and (B) mixing the specimen obtained in step (A) with a
calcium salt to prepare a measurement specimen and measuring the
clotting time of the measurement specimen.
Inventors: |
KUMANO; Osamu; (Kobe-shi,
JP) ; YAMAGUCHI; Haruki; (Kobe-shi, JP) ;
SUZUKI; Takeshi; (Kobe-shi, JP) ; IEKO; Masahiro;
(Ishikari-gun, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SYSMEX CORPORATION
SCHOOL JURIDICAL PERSON HIGASHI-NIPPON-GAKUEN |
Kobe-shi
Ishikari-gun |
|
JP
JP |
|
|
Assignee: |
SYSMEX CORPORATION
Kobe-shi
JP
SCHOOL JURIDICAL PERSON HIGASHI-NIPPON-GAKUEN
Ishikari-gun
JP
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20180031582 A1 |
February 1, 2018 |
|
|
Family ID: |
56557557 |
Appl. No.: |
15/219846 |
Filed: |
July 26, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2400/40 20130101;
G01N 33/86 20130101 |
International
Class: |
G01N 33/86 20060101
G01N033/86 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 30, 2015 |
JP |
2015-150815 |
Claims
1. A method for measuring a clotting time, comprising the steps of:
(A) mixing a blood sample, an activator, a phospholipid, and a
nickel ion-forming compound to obtain a specimen; and (B) mixing
the specimen obtained in step (A) with a calcium salt to prepare a
measurement specimen and measuring the clotting time of the
measurement specimen.
2. The method according to claim 1, wherein step (A) includes the
steps of: (A1-1) mixing a blood sample, an activator, and a
phospholipid to obtain a mixture; and (A1-2) mixing the mixture
obtained in step (A1-1) with a nickel ion-forming compound.
3. The method according to claim 1, wherein the nickel ion-forming
compound in the mixture obtained in step (A) has a final
concentration of 0.1 .mu.M or more and less than 10 mM.
4. The method according to claim 1, wherein the nickel ion-forming
compound in the mixture obtained in step (A) has a final
concentration of 0.1 mM or more and less than 5 mM.
5. The method according to claim 1, wherein, in step (B), the
mixture obtained in step (A) is incubated in a predetermined
condition, and then a calcium salt is added to the mixture.
6. The method according to claim 5, wherein the predetermined
condition is a condition of incubation at a temperature of
30.degree. C. or more and 45.degree. C. or less.
7. The method according to claim 5, wherein the predetermined
condition is a condition of incubation at a temperature of
36.degree. C. or more and 38.degree. C. or less.
8. The method according to claim 5, wherein the predetermined
condition is a condition of incubation for 1 minute or more and 6
minutes or less.
9. The method according to claim 5, wherein the predetermined
condition is a condition of incubation for 2 minutes or more and 5
minutes or less.
10. The method according to claim 5, wherein the predetermined
condition is a condition of incubation at a temperature of
30.degree. C. or more and 45.degree. C. or less for 1 minute or
more and 6 minutes or less.
11. The method according to claim 1, wherein the nickel ion-forming
compound is a compound selected from the group consisting of nickel
acetate, nickel phosphide, nickel sulfide, nickel chloride, nickel
sulfate, and nickel benzoate.
12. A measurement device for a clotting time, comprising: a
specimen preparing section that mixes a blood sample, an activator,
a phospholipid, a nickel ion-forming compound, and a calcium salt
to prepare a measurement specimen; a detection unit that obtains
clotting information showing a change associated with a clotting
reaction from the measurement specimen obtained in the specimen
preparing section; a calculator that calculates the clotting time
of the measurement specimen based on optical information obtained
by the detection unit; and a reagent accommodating portion that
accommodates an activator, a phospholipid, a nickel ion-forming
compound, and a calcium salt; wherein the specimen preparing
section obtains the activator, the phospholipid, and the nickel
ion-forming compound from the reagent accommodating portion, and
mixes the activator, the phospholipid, the nickel ion-forming
compound, and the blood sample to prepare a specimen, and the
specimen preparing section obtains the calcium salt from the
reagent accommodating portion, and mixes the calcium salt with the
specimen to obtain a measurement specimen.
13. The measurement device according to claim 12, wherein, the
specimen preparing section incubates the mixture obtained in step
(A) in a predetermined condition, and then adds a calcium salt to
the mixture.
14. The measurement device according to claim 13, wherein the
predetermined condition is a condition of incubation at a
temperature of 30.degree. C. or more and 45.degree. C. or less.
15. The measurement device according to claim 13, wherein the
predetermined condition is a condition of incubation at a
temperature of 36.degree. C. or more and 38.degree. C. or less.
16. The measurement device according to claim 13, wherein the
predetermined condition is a condition of incubation for 1 minute
or more and 6 minutes or less.
17. The measurement device according to claim 13, wherein the
predetermined condition is a condition of incubation for 2 minutes
or more and 5 minutes or less.
18. A reagent kit comprising: a first reagent containing an
activator and a phospholipid accommodated in a first reagent
container; a second reagent containing a nickel ion-forming
compound accommodated in a second reagent container; and a third
reagent containing a calcium salt accommodated in a third reagent
container.
19. The reagent kit according to claim 18, wherein the nickel
ion-forming compound is a compound selected from the group
consisting of nickel acetate, nickel phosphide, nickel sulfide,
nickel chloride, nickel sulfate, and nickel benzoate.
20. The reagent kit according to claim 18, wherein reagent kit is
used for the method of claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from prior Japanese Patent
Application No. 2015-150815, filed on Jul. 30, 2015, entitled
"METHOD FOR MEASURING CLOTTING TIME, MEASUREMENT DEVICE FOR
CLOTTING TIME, CLOTTING TIME MEASURING REAGENT, AND REAGENT KIT",
the entire contents of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to a method for measuring a
clotting time, a measurement device for a clotting time, a clotting
time measuring reagent, and a reagent kit.
BACKGROUND
[0003] Activated partial thromboplastin time (APTT) is used to
monitor the concentration of heparin as an anticoagulant (U.S. Pat.
No. 5,705,396). U.S. Pat. No. 5,705,396 describes that, with use of
an APTT measuring reagent containing a copper or zinc salt, the
activity of heparin is decreased when the APTT is measured.
[0004] However, there is a need to measure the clotting time at
high sensitivity even if a heparin-containing sample is used.
SUMMARY OF THE INVENTION
[0005] The scope of the present invention is defined solely by the
appended claims, and is not affected to any degree by the
statements within this summary.
[0006] A first aspect of the present invention includes a method
for measuring a clotting time, including the steps of:
[0007] (A) mixing a blood sample, an activator, a phospholipid, and
a nickel ion-forming compound to obtain a specimen; and
[0008] (B) mixing the specimen obtained in step (A) with a calcium
salt to prepare a measurement specimen and measuring the clotting
time of the measurement specimen.
[0009] A second aspect of the present invention includes a
measurement device for a clotting time, including: a specimen
preparing section that mixes a blood sample, an activator, a
phospholipid, a nickel ion-forming compound, and a calcium salt to
prepare a measurement specimen; a detection unit that obtains
clotting information showing a change associated with a clotting
reaction from the measurement specimen obtained in the specimen
preparing section; a calculator that calculates the clotting time
of the measurement specimen based on the optical information
obtained by the detection unit; and a reagent accommodating portion
that accommodates an activator, a phospholipid, a nickel
ion-forming compound, and a calcium salt; wherein the specimen
preparing section obtains the activator, the phospholipid, and the
nickel ion-forming compound from the reagent accommodating portion,
the activator, and mixes the activator, the phospholipid, the
nickel ion-forming compound, and the blood sample to prepare a
specimen, and the specimen preparing section obtains the calcium
salt from the reagent accommodating portion, and mixes the calcium
salt with the specimen to obtain a measurement specimen.
[0010] A third aspect of the present invention includes a clotting
time measuring reagent that is used in the method for measuring a
clotting time, which contains a nickel ion-forming compound.
[0011] A fourth aspect of the present invention includes a reagent
kit including a first reagent containing an activator and a
phospholipid accommodated in a first reagent container, a second
reagent containing a nickel ion-forming compound accommodated in a
second reagent container, and a third reagent containing a calcium
salt accommodated in a third reagent container.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a configuration diagram of a measurement device
for a clotting time;
[0013] FIG. 2 is a flow chart showing a procedure of measuring the
clotting time with a measurement device;
[0014] FIG. 3 is a flow chart showing a procedure of preparing a
specimen;
[0015] FIG. 4 is a flow chart showing a procedure of preparing a
specimen;
[0016] FIG. 5 is a flow chart showing procedures of steps of adding
a calcium salt to a specimen and obtaining optical information;
[0017] FIG. 6 is a configuration diagram of a reagent kit;
[0018] FIG. 7 is a graph showing results of comparison between the
clotting time by the measurement method of Example 1 and the
clotting time by the measurement method of Comparative Example 1;
and
[0019] FIG. 8 is a graph showing results of an examined
relationship between heparin concentration and APTT ratio in
Example 2 and Comparative Examples 2 to 5.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. Method for Measuring Clotting Time
[0020] The method for measuring a clotting time according to an
embodiment (hereinafter simply referred to as "measurement method")
includes the steps of: (A) mixing a blood sample, an activator, a
phospholipid, and a nickel ion-forming compound to obtain a
specimen; and (B) mixing the specimen obtained in step (A) with a
calcium salt to prepare a measurement specimen and measuring the
clotting time of the measurement specimen. In the measurement
method according to the embodiment, a blood sample, an activator, a
phospholipid, and a nickel ion-forming compound are mixed in
obtaining a specimen in step (A). Therefore, the measurement method
according to the embodiment allows the clotting time to be measured
at high sensitivity even if a heparin-containing sample is
used.
[0021] The term "specimen" used herein means a mixture of a blood
sample, an activator, a phospholipid, and a nickel ion-forming
compound. The term "measurement specimen" means a mixture of a
blood sample, an activator, a phospholipid, a nickel ion-forming
compound, and a calcium salt.
[0022] Examples of the blood sample include plasma, but are not
particularly limited thereto. The term "normal plasma" used herein
means plasma obtained from blood of a healthy individual. The
normal plasma may be commercially available normal plasma. Examples
of test plasma include plasma obtained from a subject, and plasma
obtained from a subject and contains heparin, but are not
particularly limited.
[0023] The activator should be a substance having an effect of
activating contact factors involved in the intrinsic coagulation
pathway. Examples of the contact factors include prekallikrein,
high-molecular-weight kininogen, and factors XII and XI, but are
not particularly limited thereto. Examples of the activator include
ellagic acid compounds, silica, kaolin, and diatomaceous earth
(e.g. product name: Celite (registered trademark), manufactured by
Celite Corporation), but are not particularly limited thereto.
These activators may be used singly, or as a mixture of two or more
kinds thereof. The term "ellagic acid compound" means a concept
including ellagic acid, and a salt and a metal complex of ellagic
acid.
[0024] A phospholipid accelerates a blood clotting reaction. The
phospholipid is a lipid having a phosphoric ester site in a
molecular structure. The phospholipid may be a naturally occurring
or synthetic phospholipid. Examples of the naturally occurring
phospholipid include phospholipids derived from animals such as
rabbit, bovine, porcine, chicken, and human; and phospholipids
derived from plants such as soybean, but are not limited thereto.
Examples of the phospholipids derived from animals include
phospholipids derived from rabbit brain, bovine brain, yolk, human
placenta, and the like, but are not limited thereto. Specific
examples of the phospholipid include glycerophospholipids such as
phosphatidylethanolamine, phosphatidylcholine, and
phosphatidylserine, but are not limited thereto. Among these
phospholipids, phosphatidylethanolamine, phosphatidylcholine, and
phosphatidylserine are preferred from the viewpoint of efficient
progression of the blood clotting reaction. These phospholipids may
be used singly, or as a mixture of two or more kinds thereof.
Examples of the fatty acid side chains in phospholipids include
palmitoyl, oleoyl, and stearoyl groups, but are not particularly
limited thereto. These fatty acid side chains may be appropriately
selected as long as the blood clotting reaction is not
hindered.
[0025] The nickel ion-forming compound should be a compound that
forms nickel ions in a blood sample. The nickel ion-forming
compound is preferably a compound that forms divalent cations.
Examples of the nickel ion-forming compound include nickel acetate,
nickel phosphide, nickel sulfide, nickel chloride, and nickel
sulfate, but are not particularly limited thereto. Among these
nickel ion-forming compounds, nickel acetate is preferred. These
nickel ion-forming compounds may be used singly, or as a mixture of
two or more kinds thereof.
[0026] The calcium salt should be a salt that forms calcium ions in
a measurement specimen. Examples of the calcium salt include
calcium chloride, calcium sulfate, calcium nitrite, calcium
carbonate, calcium lactate, and calcium tartrate, but are not
particularly limited thereto. These calcium salts may be used
singly, or as a mixture of two or more kinds thereof.
[0027] In step (A), a blood sample, an activator, a phospholipid,
and a nickel ion-forming compound are mixed to obtain a specimen.
Prior to step (A), the blood sample may be heated to a temperature
appropriate for performing the clotting reaction. Usually, the
heating temperature of the blood sample is preferably from 30 to
45.degree. C. and more preferably from 36 to 38.degree. C.
[0028] In step (A), the order of mixing the blood sample,
activator, phospholipid, and nickel ion-forming compound is not
particularly limited. Step (A) is divided into, for example, the
following aspects:
(Aspect 1)
[0029] mixing a blood sample, an activator, and a phospholipid to
obtain a mixture, and adding a nickel ion-forming compound
thereto;
(Aspect 2)
[0030] mixing a blood sample with a nickel ion-forming compound,
and adding an activator and a phospholipid thereto; and
(Aspect 3)
[0031] simultaneously adding an activator, a phospholipid, and a
nickel ion-forming compound to a blood sample.
[0032] In Aspect 1, step (A) includes, for example, the following
step (A1-1) and step (A1-2):
[0033] (A1-1) mixing a blood sample, an activator, and phospholipid
to obtain a mixture; and
[0034] (A1-2) mixing the mixture obtained in step (A1-1) with a
nickel ion-forming compound.
[0035] Hereinafter, the measurement method according to the
embodiment will be described with reference to Aspect 1, but is not
particularly limited thereto. In step (A1-1), a blood sample, an
activator, and a phospholipid are mixed to obtain a mixture. The
order of mixing the blood sample, activator, and phospholipid is
not particularly limited. The activator and phospholipid may be
mixed simultaneously with the blood sample. The activator and
phospholipid may be mixed with the blood sample at different times.
In this case, the phospholipid may be added after the activator is
added to the blood sample, or alternatively, the activator may be
added after the phospholipid is added to the blood sample.
[0036] In step (A1-1), the amount of the activator to be mixed with
the blood sample should be an amount at which the concentration of
the activator in the measurement specimen is a predetermined
concentration. The concentration of the activator in the
measurement specimen may be appropriately set depending on the type
of activator. When the activator is an ellagic acid compound,
usually, the concentration of the activator in the measurement
specimen is preferably from 3.5 to 150 .mu.M and more preferably
from 10 to 50 .mu.M. When the activator is silica, usually, the
concentration of the activator in the measurement specimen is
preferably from 0.04 to 0.4 mg/mL and more preferably from 0.07 to
0.2 mg/mL.
[0037] In step (A1-1), the amount of the phospholipid to be mixed
with the blood sample should be an amount at which the
concentration of the phospholipid in the measurement specimen is a
predetermined concentration. The concentration of the phospholipid
in the measurement specimen may be appropriately set depending on
the type of phospholipid. When the phospholipid is
phosphatidylethanolamine, usually, the concentration of the
phospholipid in the measurement specimen is preferably from 1 to
150 .mu.g/mL and more preferably from 5 to 50 .mu.g/mL. When the
phospholipid is phosphatidylcholine, usually, the concentration of
the phospholipid in the measurement specimen is preferably from 1
to 100 .mu.g/mL and more preferably from 5 to 80 .mu.g/mL. When the
phospholipid is phosphatidylserine, usually, the concentration of
the phospholipid in the measurement specimen is preferably from 0.1
to 50 .mu.g/mL and more preferably from 1 to 10 .mu.g/mL. When the
phospholipid is a mixture of two or more kinds of phospholipids,
usually, the concentration of each of the phospholipids in the
measurement specimen is preferably from 5 to 400 .mu.g/mL and more
preferably from 20 to 100 .mu.g/mL.
[0038] The heating temperature when mixing the blood sample, the
activator and/or the phospholipid should be a temperature
appropriate for performing the blood clotting reaction. Usually,
the heating temperature is preferably from 30 to 45.degree. C. and
more preferably from 36 to 38.degree. C. Usually, the heating time
is preferably from 10 to 150 seconds and more preferably from 30 to
90 seconds.
[0039] In step (A1-2), the mixture obtained in step (A1-1) is mixed
with a nickel ion-forming compound to obtain a specimen.
[0040] In step (A1-2), the amount of the nickel ion-forming
compound to be mixed with the mixture obtained in step (A1-1)
should be an amount at which the nickel ion-forming compound in the
measurement specimen has a predetermined final concentration. The
final concentration of the nickel ion-forming compound in the
measurement specimen is preferably 0.1 .mu.M or more, more
preferably 0.1 mM or more, and preferably less than 10 mM, more
preferably 5 mM or less.
[0041] The heating temperature when mixing the mixture obtained in
step (A1-1) with the nickel ion-forming compound should be a
temperature appropriate for performing the blood clotting reaction.
Usually, the temperature is preferably from 30 to 45.degree. C. and
more preferably from 36 to 38.degree. C. Usually, the heating time
is preferably from 30 to 420 seconds and more preferably from 100
to 350 seconds.
[0042] From the viewpoint of effectively preventing the clotting
time from becoming too long during the measurement of clotting
time, the mixture obtained in step (A1-1) is preferably mixed with
a nickel ion-forming compound in step (A1-2) within 150 seconds,
preferably within 60 seconds after the end of mixing the blood
sample, activator, and phospholipid in step (A1-1).
[0043] In step (B), the specimen obtained in step (A) is mixed with
a calcium salt to prepare a measurement specimen, and the clotting
time of the measurement specimen is measured.
[0044] The amount of the calcium salt to be mixed with the specimen
may be an amount at which the concentration of the calcium salt in
the measurement specimen is a predetermined concentration. The
concentration of calcium salt in the measurement specimen is
preferably from 2 to 20 mM and more preferably from 4 to 10 mM.
[0045] In step (B), the specimen may be heated to an appropriate
temperature to carry out a clotting reaction before adding the
calcium salt to the specimen. The heating temperature of the
specimen is preferably 30.degree. C. or more and more preferably
36.degree. C. or more from the viewpoint of reactivity in the
clotting reaction. The heating temperature of the specimen is
preferably 45.degree. C. or less and more preferably 38.degree. C.
or less from the viewpoint of protein stability. In this case, the
heating time is preferably 1 minute or more and more preferably 2
minutes or more from the viewpoint of reactivity in the clotting
reaction. The heating time is preferably 6 minutes or less and more
preferably 5 minutes or less from the viewpoint of protein
stability.
[0046] The clotting time of the measurement specimen can be
examined based on clotting information. Examples of the clotting
information include changes in the transmitted or scattered light
when the measurement specimen is irradiated with light and changes
in the viscosity of the measurement specimen, but are not
particularly limited thereto. In this case, the clotting time of
the measurement specimen can be examined by emitting light to the
measurement specimen, and monitoring changes in the transmitted
light passed through the measurement specimen or the scattered
light from the measurement specimen, or monitoring changes in the
viscosity of the measurement specimen. The term "clotting time"
used herein means an activated partial thromboplastin time. The
clotting time is a time from when the addition of the calcium salt
to the specimen starts till when the plasma clots.
[0047] The clotting of plasma can be determined using as an
indicator, for example, the fact that the light from the
measurement specimen irradiated with light does not change any
more, or the fact that the viscosity of the measurement specimen
does not change any more.
2. Measurement Device for Clotting Time
[Overall Configuration of Measurement Device]
[0048] An example of the measurement device for a clotting time
(hereinafter, simply referred to as "measurement device") to be
used for the measurement method as described above will be
described with reference to the attached drawings. As shown in FIG.
1, a measurement device 10 includes a measurement unit 20 and a
processing apparatus 30. The measurement unit 20 and the processing
apparatus 30 are communicably connected to each other.
[Configuration of Measurement Unit]
[0049] As shown in FIG. 1, the measurement unit 20 includes a
specimen preparing section 100, a detection unit 200, and a reagent
accommodating portion 300, a sample accommodating portion 400
accommodating a blood sample, and a controller 500.
[0050] The specimen preparing section 100 obtains a reagent from
the reagent accommodating portion 300 and also obtains a blood
sample from the sample accommodating portion 400. The specimen
preparing section 100 mixes the obtained reagent with the obtained
blood sample based on a predetermined procedure to prepare a
measurement specimen. The specimen preparing section 100 includes a
sample transporting section 111, a first reagent transporting
section 112, a second reagent transporting section 113, a third
reagent transporting section 114, a fourth reagent transporting
section 115, and a cuvette transporting section 131. The sample
transporting section 111 has a first nozzle 101. The sample
transporting section 111 obtains the blood sample accommodated in
the sample accommodating portion 400. The sample transporting
section 111 discharges the obtained blood sample into a cuvette 90.
The first reagent transporting section 112 has a second nozzle 102.
The first reagent transporting section 112 obtains a reagent
accommodated in a first container 301 of the reagent accommodating
portion 300 through the second nozzle 102. The first reagent
transporting section 112 discharges the obtained reagent into the
cuvette 90. The second reagent transporting section 113 has a third
nozzle 103. The second reagent transporting section 113 obtains a
reagent accommodated in a second container 302 of the reagent
accommodating portion 300 through the third nozzle 103. The second
reagent transporting section 113 discharges the obtained reagent
into the cuvette 90. The third reagent transporting section 114
obtains a reagent accommodated in a third container 303 of the
reagent accommodating portion 300 through a fourth nozzle 104. The
third reagent transporting section 114 discharges the obtained
reagent into the cuvette 90. The fourth reagent transporting
section 115 obtains a reagent accommodated in a fourth container
304 of the reagent accommodating portion 300 through a fifth nozzle
105. The fourth reagent transporting section 115 discharges the
obtained reagent into the cuvette 90. The cuvette transporting
section 131 transports the cuvette 90 accommodating a prepared
measurement specimen to the detection unit 200.
[0051] The detection unit 200 includes a light irradiation unit
201, a light receiver 202, and a second cuvette mounting portion
203. The light irradiation unit 201 has a light source of light
emitted to a measurement specimen. The wavelength of emitted light
should be a wavelength suitable for monitoring the change with the
progress of the clotting reaction of blood. The light receiver 202
receives light from the measurement specimen. The light from the
measurement specimen may be transmitted or scattered light. The
light receiver 202 outputs an electric signal corresponding to the
amount of the received light to a calculator 31 of the processing
apparatus. The second cuvette mounting portion 203 is provided
between the light irradiation unit 201 and the light receiver 202.
The cuvette 90 transported from the specimen preparing section 100
is placed in the second cuvette mounting portion 203.
[0052] The reagent accommodating portion 300 accommodates a reagent
used for measurement of clotting time. In the embodiment, the
reagent accommodating portion 300 includes a first container 301
that accommodates an activator, a second container 302 that
accommodates a phospholipid, a third container 303 that
accommodates a nickel ion-forming compound, and a fourth container
304 that accommodates a calcium salt. An identifier for identifying
the kind of reagent accommodated in the container is provided in
each of the first to fourth containers. Examples of the identifiers
include bar codes, but are not particularly limited thereto. In the
embodiment, the first container 301 and the second container 302 as
different containers are provided in the reagent accommodating
portion 300. However, since the activator and phospholipid may be
simultaneously mixed with a blood sample, the activator and
phospholipid may be accommodated in one common container in place
of the first and second containers. In this case, the first reagent
transporting section 112 and the second reagent transporting
section 113 are one common transporting section.
[0053] The sample accommodating portion 400 accommodates a blood
sample. In the embodiment, the sample accommodating portion 400
includes a plurality of sample containers 401. The sample
accommodating portion 400 transports the sample containers 401
accommodating desired blood samples to a predetermined sample
aspirating position. Identifiers for identifying the kinds of blood
samples accommodated in the containers are provided in the sample
containers 401. Examples of the identifiers include bar codes, but
are not particularly limited thereto.
[0054] The controller 500 includes a central processing unit (CPU)
501 and a storage unit 502. The controller 500 is composed of a
computer. The CPU 501 executes the computer program stored in the
storage unit 502. Thus, the CPU 501 prepares the specimen in the
specimen preparing section 100 and provides optical information on
a measurement specimen in the detection unit 200. Examples of the
computer program include a computer program for preparing a
measurement specimen and a computer program for providing optical
information on the measurement specimen, but are not particularly
limited thereto. The storage unit 502 further stores reagent
identification information to identify a reagent accommodated in
the reagent accommodating portion 300, specimen preparation
information on procedures in preparing a measurement specimen, and
sample identification information to identify a blood sample
accommodated in the sample accommodating portion 400. Examples of
the reagent identification information include information on
association of the type of reagent, the position of accommodating
containers, and identifiers, but are not particularly limited
thereto. Examples of the sample identification information include
information on association of the type of blood sample, the
position of accommodating containers, and identifiers, but are not
particularly limited thereto. The CPU 501 executes the computer
program for preparing a measurement specimen using the reagent
identification information and specimen preparation information
stored in the storage unit 502. Thus, the CPU 501 makes the
specimen preparing section 100 of the measurement unit 20 prepare
the measurement specimen.
[Configuration of Processing Apparatus]
[0055] As shown in FIG. 1, the processing apparatus 30 includes a
calculator 31, a display unit 32, and an input unit 33. In the
embodiment, the processing apparatus 30 is composed of a computer
system. The calculator 31 includes a CPU 601 and a storage unit
602. The CPU 601 executes the computer program stored in the
storage unit 602. Thus, the CPU 601 calculates clotting time.
Examples of the display unit 32 include screen displays, but are
not particularly limited thereto. The display unit 32 displays, for
example, information on the calculated clotting time. Examples of
the input unit 33 include keyboards and mice, but are not
particularly limited thereto.
[0056] The storage unit 602 is installed with computer programs to
be executed by the CPU 601, such as an operating system and an
application program, as well as data used in executing the computer
programs. Examples of the application program include computer
programs for measuring the clotting time, but are not particularly
limited thereto. The CPU 601 executes the computer program to
measure clotting time stored in the storage unit 602. Thus, the CPU
601 makes the measurement device 10 measure clotting time.
[Modification of Measurement Device]
[0057] The sample transporting section 111, the first reagent
transporting section 112, the second reagent transporting section
113, the third reagent transporting section 114, and the fourth
reagent transporting section 115 may each be a flow path for
flowing a sample or reagent. Examples of the flow paths include
tubes, but are not particularly limited thereto.
[0058] The clotting time may be measured based on the increase in
the viscosity due to blood clotting and other clotting information.
When the clotting time is measured based on the increase in the
viscosity due to blood clotting, the detection unit 200 includes a
high frequency transmitting coil, a high frequency receiving coil,
a cuvette mounting portion which is located between the high
frequency transmitting coil and the high frequency receiving coil
on which a cuvette accommodating a steel ball is mounted, and
electromagnets provided at both ends of the cuvette mounting
portion. The steel ball in the cuvette vibrates from side to side
due to the magnetism generated by the electromagnets. The amplitude
of vibration decreases as the viscosity increases. When the
clotting of the measurement specimen starts, the viscosity of the
measurement specimen increases, whereby the amplitude of the steel
ball decreases. Therefore, the detection unit 200 detects changes
in amplitude based on reception of a high-frequency wave
transmitted by the high frequency transmitting coil by a high
frequency receiving coil. The calculator 31 of the processing
apparatus 30 calculates clotting time based on the detected changes
in amplitude.
[Procedure of Measuring Clotting Time by Measurement Device]
[0059] Subsequently, an overview of a procedure of measuring the
clotting time by the measurement device 10 will be described with
reference to FIG. 2. In the following procedure, the controller 500
of the measurement unit 20 executes the computer program for
preparing a measurement specimen which is stored in the storage
unit 502 using the reagent identification information and specimen
preparation information obtained from the storage unit 502. The
controller 500 executes the computer program for providing optical
information on the measurement specimen which is stored in the
storage unit 502. The calculator 31 of the processing apparatus 30
executes the computer program for measuring the clotting time which
is stored in the storage unit 602 using the obtained optical
information.
[0060] In Step S1, the controller 500 of the measurement unit 20
makes the specimen preparing section 100 prepare a specimen. The
specimen preparation in Step S1 is executed in accordance with the
following procedures shown in FIGS. 3 and 4.
[0061] Thereafter, in Step S2, the controller 500 makes the
specimen preparing section 100 add a calcium salt to the specimen.
In Step S3, the controller 500 makes the detection unit 200 provide
optical information on the measurement specimen. The addition of
the calcium salt to the specimen in Step S2 and the provision of
the optical information in Step S3 are executed in accordance with
the procedure shown in FIG. 5.
[0062] Thereafter, in Step S4, the calculator 31 of the processing
apparatus 30 executes a computer program for calculating clotting
time to calculate the clotting time.
[Procedure of Preparing Specimen]
[0063] Subsequently, an overview of a procedure of preparing a
specimen by the measurement device 10 will be described with
reference to FIGS. 3 and 4.
[0064] In Step S101, the controller 500 first makes the specimen
preparing section 100 place the cuvette 90 in a specimen preparing
position 62 in FIG. 1. Specifically, the controller 500 makes the
specimen preparing section 100 mount the cuvette 90 in a first
cuvette mounting portion 61 in FIG. 1. Thus, the cuvette 90 is
placed in the specimen preparing position 62.
[0065] Then, in Step S102, the controller 500 makes the sample
accommodating portion 400 transport the sample container 401 to the
sample aspirating position 81 in FIG. 1. At this time, the
controller 500 makes the sample accommodating portion 400 select a
sample container 401 accommodating a desired blood sample based on
the sample identification information stored in the storage unit
502. Then, the controller makes the sample accommodating portion
400 transport the selected sample container 401 so as to be located
in the sample aspirating position 81.
[0066] Then, in Step S103, the controller 500 makes the specimen
preparing section 100 transfer the first nozzle 101 to the sample
aspirating position 81. Thereafter, in Step S104, the controller
500 makes the specimen preparing section 100 aspirate the blood
sample from the sample container 401. Specifically, the controller
500 makes the specimen preparing section 100 aspirate the blood
sample accommodated in the sample container 401 through the first
nozzle 101.
[0067] Then, in Step S105, the controller 500 makes the specimen
preparing section 100 transfer the first nozzle 101 to the specimen
preparing position 62. Thereafter, in Step S106, the controller 500
makes the specimen preparing section 100 discharge the blood sample
to the cuvette 90. Specifically, the controller 500 makes the
specimen preparing section 100 discharge the blood sample aspirated
by the first nozzle 101 to the cuvette 90.
[0068] Then, in Step S107, the controller 500 makes the specimen
preparing section 100 transfer the second nozzle 102 to an
activator aspirating position 71. Thereafter, in Step S108, the
controller 500 makes the specimen preparing section 100 aspirate an
activator from the first container 301. Specifically, the
controller 500 makes the specimen preparing section 100 aspirate
the activator accommodated in the first container 301 through the
second nozzle 102.
[0069] Then, in Step S109, the controller 500 makes the specimen
preparing section 100 transfer the second nozzle 102 to the
specimen preparing position 62. Thereafter, in Step S110, the
controller 500 makes the specimen preparing section 100 discharge
the activator to the cuvette 90. Specifically, the controller 500
makes the specimen preparing section 100 discharge the activator
aspirated through the second nozzle 102 to the cuvette 90.
[0070] Then, in Steps S111 to S114 of FIG. 4, the controller 500
makes the specimen preparing section 100 transfer the third nozzle
103 to the phospholipid aspirating position 72, aspirate a
phospholipid from the second container 302, transfer the third
nozzle 103 to the specimen preparing position 62, and discharge the
phospholipid to the cuvette 90, respectively. Steps S111 to S114
are respectively the same as Steps S107 to S110 of FIG. 3 except
for a series of operations including aspirating and discharging the
phospholipid through the third nozzle 103.
[0071] Then, in Steps S115 to S118, the controller 500 makes the
specimen preparing section 100 transfer the fourth nozzle 104 to a
nickel ion-forming compound aspirating position 73, aspirate the
nickel ion-forming compound from the third container 303, transfer
the fourth nozzle 104 to the specimen preparing position 62, and
discharge the nickel ion-forming compound to the cuvette 90,
respectively. Steps S115 to S118 are respectively the same as Steps
S107 to S110 of FIG. 3 except for a series of operations including
aspirating and discharging the nickel ion-forming compound through
the fourth nozzle 104. As a result, a specimen is obtained.
[0072] In the embodiment, the activator and phospholipid are added
to the blood sample in this order. However, the activator and
phospholipid may be simultaneously added thereto.
[Procedures of Adding Calcium Salt to Specimen and Obtaining
Optical Information]
[0073] Subsequently, an overview of procedures of adding a calcium
salt to a specimen by the measurement device 10 and obtaining
optical information will be described with reference to FIG. 5.
[0074] In Steps S201 to S204 of FIG. 5, the controller 500 makes
the specimen preparing section 100 transfer the fifth nozzle 105 to
a calcium salt aspirating position 74, aspirate the calcium salt
from the second container 302, transfer the fifth nozzle 105 to the
specimen preparing position 62, and discharge the calcium salt to
the cuvette 90, respectively. As a result, a measurement specimen
is obtained. Steps S201 to S204 are respectively the same as Steps
S107 to S110 of FIG. 3 except for a series of operations including
aspirating and discharging the calcium salt through the fifth
nozzle 105.
[0075] In Step S301, simultaneously with Step S204, the controller
500 makes the specimen preparing section 100 transfer the cuvette
90 to the second cuvette mounting portion 203 of the detection unit
200 through the cuvette transporting section 131. In Step S301, the
controller 500 makes the detection unit 200 irradiate the
measurement specimen with light. Specifically, the controller 500
makes the light irradiation unit 201 of the detection unit 200 emit
light to the cuvette 90 mounted in the second cuvette mounting
portion 203. As a result, the measurement specimen in the cuvette
90 is irradiated with light.
[0076] Then, in Step 302, the controller 500 makes the detection
unit 200 measure the light from the measurement specimen.
Specifically, the controller 500 makes the calculator 31 of the
processing apparatus 30 output an electric signal corresponding to
the amount of the transmitted light received by the light receiver
202 of the detection unit 200.
[0077] Thereafter, the process proceeds to the calculation of
clotting time in Step S4 of FIG. 2.
[Modification of Operation Procedures]
[0078] A series of Steps S107 to S110 may be performed in
conjunction with a series of Steps S111 to S114. A series of Steps
S115 to S118 may be performed before both of the series of Steps
S107 to S110 and the series of Steps S111 to S114.
[0079] When the clotting time is measured based on the increase in
the viscosity due to blood clotting, one usable as the detection
unit 200 is a detection unit that includes a high frequency
transmitting coil, a high frequency receiving coil, a cuvette
mounting portion on which a cuvette accommodating a steel ball is
mounted, and an electromagnet. Here, the controller 500 makes the
detection unit 200 detect changes in amplitude based on reception
of a high-frequency wave transmitted by the high frequency
transmitting coil of the detection unit 200 by the high frequency
receiving coil. Then, the controller 500 makes the processing
apparatus 30 output information on changes in amplitude detected by
the detection unit 200. Thereafter, the calculator 31 of the
processing apparatus 30 uses the obtained information on changes in
amplitude, and executes the computer program for measuring the
clotting time which is stored in the storage unit 602 to calculate
the clotting time.
3. Clotting Time Measuring Reagent
[0080] The clotting time measuring reagent according to the
embodiment is a clotting time measuring reagent used in the method
for measuring a clotting time which contains a nickel ion-forming
compound. The nickel ion-forming compound is the same as the nickel
ion-forming compound in the measurement method.
[0081] The clotting time measuring reagent according to the
embodiment may be a reagent that is substantially formed of a
nickel ion-forming compound, or a reagent that contains a nickel
ion-forming compound, an appropriate solvent, and further an
adjuvant. The clotting time measuring reagent according to the
embodiment does not substantially contain a phospholipid and an
activator.
[0082] The clotting time measuring reagent may be provided in a
solid state. In this case, examples of dosage forms of the clotting
time measuring reagent include granules and dust formulations, but
are not particularly limited thereto.
[0083] The clotting time measuring reagent may be in a state where
the nickel ion-forming compound is dissolved in an appropriate
solvent. In this case, examples of the solvent include desalinated
and purified water and physiological saline, but are not
particularly limited thereto.
[0084] When the clotting time measuring reagent is a reagent in a
state where the nickel ion-forming compound is dissolved in an
appropriate solvent, the content of the nickel ion-forming compound
in the clotting time measuring reagent is preferably 1 .mu.M or
more, more preferably 0.1 mM or more, and preferably 50 mM or less,
more preferably 10 mM or less.
[0085] When the clotting time measuring reagent further contains an
adjuvant, examples of the adjuvant include a stabilizer and a
preservative for the nickel ion-forming compound, but are not
particularly limited thereto.
4. Reagent Kit
[0086] The reagent kit according to the embodiment is a reagent kit
including a first reagent containing an activator and a
phospholipid accommodated in a first reagent container, a second
reagent containing a nickel ion-forming compound accommodated in a
second reagent container, and a third reagent containing a calcium
salt accommodated in a third reagent container. An example of the
reagent kit according to the embodiment is a reagent kit 800 shown
in FIG. 6, but is not particularly limited thereto. The reagent kit
800 shown in FIG. 6 includes a first reagent container 801, a
second reagent container 802, and a third reagent container 803.
The first reagent container 801 accommodates the first reagent
containing an activator and a phospholipid. The second reagent
container 802 accommodates the second reagent containing a nickel
ion-forming compound. The third reagent container 803 accommodates
the third reagent containing a calcium salt. The reagent kit may
further include a package insert. The package insert may include
the description of a procedure to perform the method for measuring
a clotting time using the reagent kit according to the
embodiment.
[0087] The concentration of the activator in the first reagent
should be within a range in which the concentration in the
measurement specimen can be adjusted in the range of the
concentration in the measurement method. When the activator is an
ellagic acid compound, usually, the concentration of the activator
in the first reagent is preferably from 10 to 400 .mu.M and more
preferably from 50 to 150 .mu.M. When the activator is silica,
usually, the concentration of the activator in the first reagent is
preferably from 0.1 to 1 mg/mL and more preferably from 0.2 to 0.6
mg/mL.
[0088] The concentration of the phospholipid in the first reagent
should be within a range in which the concentration in the
measurement specimen can be adjusted in the range of the
concentration in the measurement method. Usually, the concentration
of the phospholipid in the first reagent is preferably from 30 to
400 .mu.g/mL and more preferably from 10 to 100 .mu.g/mL. When the
phospholipid is phosphatidylethanolamine, usually, the
concentration of the phospholipid in the first reagent is
preferably from 10 to 100 .mu.g/mL and more preferably from 20 to
50 .mu.g/mL. When the phospholipid is phosphatidylcholine, usually,
the concentration of the phospholipid in the measurement specimen
is preferably from 10 to 300 .mu.g/mL and more preferably from 10
to 100 .mu.g/mL. When the phospholipid is phosphatidylserine,
usually, the concentration of the phospholipid in the measurement
specimen is preferably from 1 to 75 .mu.g/mL and more preferably
from 2 to 15 .mu.g/mL.
[0089] The second reagent may be a nickel ion-forming compound in a
solid state, or may be in a state where a nickel ion-forming
compound is dissolved in an appropriate solvent. The solvent is the
same as the solvent in the clotting time measuring reagent.
[0090] When the second reagent is a reagent in a state where a
nickel ion-forming compound is dissolved in an appropriate solvent,
the concentration of the nickel ion-forming compound in the second
reagent and the concentration of the phospholipid in the first
reagent should be within a range in which the concentration in the
measurement specimen can be adjusted in the range of the
concentration in the measurement method. In this case, the
concentration of the nickel ion-forming compound in the second
reagent is preferably 1 .mu.M or more, more preferably 0.1 mM or
more, and preferably 50 mM or less, more preferably 10 mM or
less.
[0091] The concentration of the calcium salt in the third reagent
should be within a range in which the concentration in the
measurement specimen can be adjusted in the range of the
concentration in the measurement method. The concentration of the
calcium salt in the third reagent is preferably from 2.5 to 40 mM
and more preferably from 10 to 30 mM.
[0092] In the reagent kit according to the embodiment, the second
reagent does not substantially contain the phospholipid and
activator. In the reagent kit according to the embodiment, the
first reagent does not substantially contain the nickel ion-forming
compound.
[0093] The activator, phospholipid, nickel ion-forming compound,
and calcium salt used in the reagent kit are the same as those used
in the measurement method. Each of the reagent containers may
accommodate an appropriate solvent, an adjuvant or the like, if
appropriate. The solvent and adjuvant are the same as the solvent
and reagent used in the clotting time measuring reagent. In the
reagent kit according to the embodiment, the activator and
phospholipid may be accommodated in separate containers.
EXAMPLES
[0094] The clotting time was measured with a fully automated
clotting time measurement device (product name: CS-2000i,
manufactured by Sysmex Corporation).
Example 1
[0095] In this example, normal plasma or test plasma was used as a
blood sample. The used normal plasma is the normal plasma shown in
Table 1. The used test plasma is the heparin-containing plasma
shown in Table 1.
TABLE-US-00001 TABLE 1 Blood sample LotA1156, manufactured by
Precision Normal plasma Pooled Normal Plasma (PBI) BioLogic
Incorporated Test Heparin- HE1 Specimen containing Addition of novo
heparin to control plasma Mixed with Pooled Normal plasma
containing 0.1 U/mL novo heparin (product name: Coagtrol Lot. 022,
Plasma (PBI) at 1:1 plasma manufactured by Sysmex Corporation)
Heparin- HE2 Specimen containing Addition of novo heparin to
control plasma containing 0.1 U/mL novo heparin (product name:
Coagtrol Lot. 022, plasma manufactured by Sysmex Corporation)
Heparin- HE3 Specimen containing Addition of novo heparin to
control plasma Mixed with Pooled Normal containing 0.2 U/mL novo
heparin (product name: Coagtrol Lot. 022, Plasma (PBI) at 1:1
plasma manufactured by Sysmex Corporation) Heparin- HE4 Specimen
containing Addition of novo heparin to control plasma containing
0.2 U/mL novo heparin (product name: Coagtrol Lot. 022, plasma
manufactured by Sysmex Corporation) Heparin- HE5 Specimen
containing Addition of novo heparin to control plasma Mixed with
Pooled Normal containing 0.3 U/mL novo heparin (product name:
Coagtrol Lot. 022, Plasma (PBI) at 1:1 plasma manufactured by
Sysmex Corporation) Heparin- HE6 Specimen containing Addition of
novo heparin to control plasma containing 0.3 U/mL novo heparin
(product name: Coagtrol Lot. 022, plasma manufactured by Sysmex
Corporation) Heparin- HE7 Sample containing Addition of novo
heparin to control plasma Mixed with Pooled Normal containing 0.4
U/mL novo heparin (product name: Coagtrol Lot. 022, Plasma (PBI) at
1:1 plasma manufactured by Sysmex Corporation) Heparin- HE8
Specimen containing Addition of novo heparin to control plasma
containing 0.4 U/mL novo heparin (product name: Coagtrol Lot. 022,
plasma manufactured by Sysmex Corporation) Heparin- HE9 Specimen
containing Addition of novo heparin to control plasma Mixed with
Pooled Normal containing 0.6 U/mL novo heparin (product name:
Coagtrol Lot. 022, Plasma (PBI) at 1:1 plasma manufactured by
Sysmex Corporation) Heparin- HE10 Specimen containing Addition of
novo heparin to control plasma containing 0.6 U/mL novo heparin
(product name: Coagtrol Lot. 022, plasma manufactured by Sysmex
Corporation)
[0096] First, 50 .mu.L of a blood sample was heated at 37.degree.
C. for 60 seconds. Then, 50 .mu.L of an APTT reagent (product name:
PTT-LA (registered trademark), manufactured by Roche Diagnostics
K.K.) was added to the heated blood sample and mixed therewith. The
obtained mixture was heated at 37.degree. C. for 20 seconds. Then,
20 .mu.L of a 2.5 mM aqueous nickel acetate solution was added to
the heated mixture and mixed therewith. The obtained mixture was
heated at 37.degree. C. for 170 seconds. As a clotting reaction
accelerator, a 25 mM aqueous calcium chloride solution was added to
the heated mixture and the clotting time was measured. Clotting
times measured by the measurement method of Example 1 are shown in
FIG. 7.
Comparative Example 1
[0097] In this comparative example, the used blood sample is the
same blood sample as in Example 1. First, 50 .mu.L of the blood
sample was heated at 37.degree. C. for 60 seconds. Then, 50 .mu.L
of an APTT reagent (product name: PTT-LA (registered trademark),
manufactured by Roche Diagnostics K.K.) was added to the heated
blood sample and mixed therewith. The obtained mixture was heated
at 37.degree. C. for 170 seconds. A 25 mM aqueous calcium chloride
solution was added to the heated mixture and the clotting time was
measured. Clotting times measured by the measurement method of
Comparative Example 1 are shown in FIG. 7.
(Results)
[0098] The results in FIG. 7 show that the clotting time measured
by the measurement method of Example 1 is longer than the clotting
time measured by the measurement method of Comparative Example 1.
Therefore, it is found that the measurement method of Example 1
allows the clotting time to be measured at high sensitivity,
compared to the measurement method of Comparative Example 1, even
if heparin-containing sample plasma is used.
Example 2 and Comparative Examples 2 to 5
[0099] Operations were performed in the same manner as in Example 1
except that the normal plasma shown in Table 1 as blood samples and
HE2, HE4, HE6, HE8, and HE10 of the test plasma shown in Table 1
were used, and then the clotting times were measured (Example 2).
The clotting time obtained by the measurement method of Example 2
was used, and the APTT ratio was calculated in accordance with
Formula (I):
(APTT ratio)=(clotting time of test plasma/clotting time of normal
plasma) (I).
[0100] The operations was performed in the same manner as in
Example 1 except that a 2.5 mM aqueous calcium chloride solution
(Comparative Example 2), a 2.5 mM aqueous magnesium chloride
solution (Comparative Example 3), a 2.5 mM aqueous copper sulfate
solution (Comparative Example 4), and a 2.5 mM aqueous zinc
chloride solution (Comparative Example 5) were used in place of a
2.5 mM aqueous nickel acetate solution, and the normal plasma shown
in Table 1 as blood samples and HE2, HE4, HE6, HE8, and HE10 of the
test plasma shown in Table 1 were used, and then the clotting time
was measured. Calcium chloride, magnesium chloride, copper sulfate,
and zinc chloride are compounds that form divalent ions (divalent
cations) other than divalent nickel ions.
[0101] The clotting times obtained by the measurement methods of
Comparative Examples 1 to 5 were used and the APTT ratios were
measured in accordance with Formula (I). FIG. 8 shows the results
of the examined relationship between heparin concentration and APTT
ratio. In the graph, a white circle represents the APTT ratio
measured by the measurement method of Example 2 (in the presence of
the nickel ion-forming compound), a black square represents the
APTT ratio measured by the measurement method of Comparative
Example 1 (in the absence of the compound that forms divalent
cations), a white triangle represents the APTT ratio measured by
the measurement method of Comparative Example 2 (in the presence of
the compound that forms calcium ions), a black triangle represents
the APTT ratio measured by the measurement method of Comparative
Example 3 (in the presence of the compound that forms magnesium
ions), a white square represents the APTT ratio measured by the
measurement method of Comparative Example 4 (in the presence of the
compound that forms copper ions), and a black dot represents the
APTT ratio measured by the measurement method of Comparative
Example 5 (in the presence of the compound that forms zinc
ions).
[0102] The results shown in FIG. 8 show that the APTT ratio
measured by the measurement method of Example 2 is larger than the
APTT ratios measured by the measurement methods of Comparative
Examples 1 to 5. Therefore, as in Example 2, it is found that when
the nickel ion-forming compound is used, the sensitivity to the
heparin-containing sample is improved, compared to the case of
using other compounds that forms divalent ions (Comparative
Examples 2 to 5) and the case of the conventional method
(Comparative Example 1). On the other hand, as in the case of the
measurement methods of Comparative Examples 2 to 5, it is found
that when other compounds that form divalent ions are used, the
APTT ratio is equal to or lower than the APTT ratio measured by the
measurement method of Comparative Example 1.
[0103] These results show that, in the clotting time measurement
using the APTT reagent, the clotting time can be measured at high
sensitivity by adding a compound that forms nickel ions among
divalent cations to a blood sample before adding a clotting
reaction accelerator.
* * * * *