U.S. patent application number 11/841684 was filed with the patent office on 2008-02-14 for compositions, kit and one-step method for monitoring compounds having anti-factor xa and/or anti factor iia activities.
Invention is credited to Thye Yin.
Application Number | 20080038764 11/841684 |
Document ID | / |
Family ID | 35394745 |
Filed Date | 2008-02-14 |
United States Patent
Application |
20080038764 |
Kind Code |
A1 |
Yin; Thye |
February 14, 2008 |
COMPOSITIONS, KIT AND ONE-STEP METHOD FOR MONITORING COMPOUNDS
HAVING ANTI-FACTOR Xa AND/OR ANTI FACTOR IIa ACTIVITIES
Abstract
Provided are compositions and methods for accurate determination
of the concentration of anticoagulant in a sample such as a blood
or plasma sample. The compositions can contain a Factor X compound
and Factor V compound, and additional components as well. Methods
for performing the assay include one-step methods in which a sample
is added to a coagulation assay composition, and time is monitored
from the point of adding the sample and coagulation assay
composition to an endpoint, such as clot formation.
Inventors: |
Yin; Thye; (St. Louis,
MO) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
35394745 |
Appl. No.: |
11/841684 |
Filed: |
August 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11127540 |
May 11, 2005 |
7294479 |
|
|
11841684 |
Aug 20, 2007 |
|
|
|
60570312 |
May 11, 2004 |
|
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Current U.S.
Class: |
435/13 ;
435/287.1 |
Current CPC
Class: |
G01N 2333/96444
20130101; C12Q 1/56 20130101; G01N 33/86 20130101; G01N 2333/7456
20130101 |
Class at
Publication: |
435/013 ;
435/287.1 |
International
Class: |
C12Q 1/56 20060101
C12Q001/56; C12M 1/00 20060101 C12M001/00 |
Claims
1. A blood coagulation assay composition, comprising: (a) a
partially or substantially pure Factor X compound; (b) Factor V
compound; and (c) a multivalent cation, wherein the components of
the composition are substantially unreacted with one another.
2. The composition of claim 1, wherein the composition is
substantially free of liquid or gaseous H.sub.2O.
3. The composition of claim 1, wherein the composition is
substantially free of products of a clotting reaction.
4. The composition of claim 1, further characterized by providing a
linear anticoagulant curve when anticoagulant concentration is
plotted versus clotting time.
5. The composition of claim 1, further comprising a calcium
salt.
6. The composition of claim 1, further comprising lipid or
detergent.
7. The composition of claim 1, wherein Factor V compound is a
component of a plasma fraction.
8. The composition of claim 1, further comprising fibrinogen.
9. The composition of claim 1, further comprising a plasma fraction
containing Factor V compound, and further containing fibrinogen and
lipid or detergent added thereto.
10. The composition of claim 1, further comprising a plasma
fraction containing Factor V compound, and further containing
prothrombin complex, anticoagulant, lipid or detergent, and normal
plasma added thereto.
11. The composition of claim 1, wherein the composition has been
lyophilized.
12. The composition of claim 1, wherein the composition provides a
linear correlation between clotting time and heparin concentration
range, wherein the heparin concentration range is selected from the
group consisting of 0 units/mL to about 20 units/mL, 0 units/mL to
about 15 units/mL, 0 units/mL to about 12 units/mL, 0 units/mL to
about 10 units/mL, 0 units/mL to about 7.5 units/mL, and 0 units/mL
to about 5 units/mL.
13. The composition of claim 1, wherein the Factor X compound is
Factor X.sub.a.
14. The composition of claim 1, wherein the Factor V compound is
Factor V.
15. The composition of claim 1, further comprising benzamidine.
16. The composition of claim 1, wherein Factor X compound is
present in excess relative to the amount of sample to be added to
the composition.
17. The composition of claim 1, wherein Factor V compound is
present in excess relative to the amount of sample to be added to
the composition.
18. A blood coagulation assay composition, comprising: (a) a Factor
X compound; and (b) Factor V compound, wherein said Factor X
compound and said Factor V compound are substantially not
admixed.
19. A combination comprising the composition of claim 1 located in
a single chamber vessel.
20. The combination of claim 19, wherein the vessel is sealed with
a vapor-impermeable seal.
21. The combination of claim 19, wherein a steel ball is located
within the vessel.
22. A kit, comprising the composition of claim 19, further
including a reference for quantitating the results of a coagulation
assay.
23. A method for preparing the blood coagulation assay composition
of claim 1, comprising: (a) placing Factor X compound into a
vessel; and (b) placing Factor V compound into the vessel, whereby
said Factor X compound and said Factor V compound are substantially
not admixed, thereby forming the composition of claim 1.
24. A method of assaying coagulation of a sample, comprising: (a)
mixing a sample with the coagulation assay composition of claim 1;
and (b) determining the time period beginning upon performing step
(a) and ending upon clot formation.
25. A method for preparing a blood coagulation assay composition,
comprising: (a) placing Factor X compound into a vessel; and (b)
placing Factor V compound into the vessel; whereby said Factor X
compound and said Factor V compound are substantially not
admixed.
26. A method of assaying coagulation of a sample, comprising: (a)
mixing a sample with a coagulation assay composition, wherein
Factor X compound and Factor V compound in said coagulation assay
composition are substantially not admixed prior to said mixing of
the sample with the coagulation assay composition; and (b)
determining the time period beginning upon performing step (a) and
ending upon clot formation.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 11/127,540, filed May 11, 2005, which claims priority under 35
U.S.C. .sctn.119(e) to U.S. Provisional Application Ser. No.
60/570,312, filed on May 11, 2004, to Thye Yin. The subject matter
of the above-noted patent applications are herein incorporated in
their entirety.
BACKGROUND
[0002] 1. Technical Field
[0003] The claimed subject matter relates to testing blood clotting
in blood, and more particularly to determining the anti-Factor
X.sub.a and/or anti Factor II.sub.a activities of compounds in a
blood sample.
[0004] 2. Background
[0005] The determination of heparin establishes an important
parameter for the supervision of heparin treatment, which is often
administered in the presence of a threat of thrombosis. Heparin
forms with antithrombin III (AT III) a complex that inhibits the
proteolytic activity of FX.sub.a and thrombin. Heparin treatment is
often applied in the presence of a threat of thrombosis (e.g.
before surgical interventions). Adjustment of heparin concentration
is therefore important. If the dose is too low, there is the danger
of thrombosis or embolism, which can result in death. Excessively
high heparin concentrations, however, result in bleeding. The
quantitative analysis of heparin, therefore, is one of the tests
most frequently performed in a blood testing laboratory.
[0006] In 1973 Yin and co-workers developed the first quantitative
assay method for the in vitro measurement of heparin in plasma
based on Factor X.sub.a neutralization (J. Lab. Clin. Med. 81:298,
1973). This assay method could detect less than 0.02 units of
heparin per milliliter of plasma. However, the assay is cumbersome
and time consuming to perform. It is a two-stage assay, and
requires much manual manipulation. In the first-stage of the assay,
the patient's plasma sample is incubated with the sample plasma,
buffer, and a known excess of Factor X.sub.a for a predetermined
time period, after which a sub sample from this primary reaction
mixture is removed and assayed for residual Factor X.sub.a
activity. The latter step constitutes the second-stage of the
assay. The residual Factor X.sub.a activity is measured by the
addition of the test sample to another test tube, and to it calcium
chloride, cephalin in bovine plasma are added separately in timed
fashion. Other assays that presently exist are either cumbersome or
limited in scope of application. Therefore, a need exists for
improved heparin assay methods.
SUMMARY
[0007] The details of one or more embodiments of the invention are
set forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and drawings, and from the claims.
[0008] Provided herein are blood coagulation assay compositions
that contain a Factor X compound and a Factor V compound. The
compositions can also include a variety of additional components
such as a multivalent cation, lipid or detergent, fibrinogen,
anticoagulant, prothrombin complex, normal plasma. In one
embodiment, the Factor V compound is a component of a plasma
fraction. The compositions can be present in a vessel substantially
free of liquid or gaseous H.sub.2O. The compositions can be present
in a vessel in substantially unreacted or unactivated form.
[0009] Also provided herein are methods for preparing a blood
coagulation assay composition by placing Factor X compound into a
vessel and placing Factor V compound into the vessel in such a
manner that the components do not substantially react. In one
embodiment, the components are added in such a manner that any
liquid placed into the vessel is flash frozen upon placement into
the vessel.
[0010] Also provided herein are methods of assaying coagulation of
a sample by mixing a sample with a coagulation assay composition
and determining the time period beginning upon mixing the sample
and coagulation assay composition and ending upon clot formation.
In one embodiment, only a single mixing step is required.
[0011] Provided herein are blood coagulation assay compositions
that contain a Factor X compound and Factor V compound. The
compositions provided herein can be substantially free of liquid or
gaseous H.sub.2O. The compositions provided herein can contain
components of the composition that are substantially unreacted with
one another. The compositions provided herein can be substantially
free of products of a clotting reaction. In the compositions
provided herein, Factor X compound can be substantially free of
antithrombin III bound thereto. In the compositions provided
herein, addition of blood or a blood sample to the composition can
initiate a clotting reaction. In the compositions provided herein,
substantially all of the phospholipid of the composition can be
aqueously dispersed. In the compositions provided herein,
substantially all of the calcium salt of the composition can be
aqueously soluble. In the compositions provided herein,
substantially all of the Factor Xa of the composition can be
aqueously soluble. In the compositions provided herein,
substantially all of the Factor Xa of the composition can be
unbound by antithrombin III. The compositions provided herein can
be further characterized by providing a linear anticoagulant curve
when anticoagulant concentration is plotted versus clotting time.
In the compositions provided herein, substantially all of the
Factor V compound of the composition can be aqueously soluble. In
the compositions provided herein, substantially all components of
the composition can be aqueously soluble. In the compositions
provided herein, the composition can be substantially free of
Factor IIa. In the compositions provided herein, the composition
can be substantially free of fibrin.
[0012] The compositions provided herein can further contain a
multivalent cation. In the compositions provided herein, the
multivalent cation can be selected from the group consisting of
Mg.sup.2+ ion and Ca.sup.2+ ion. In the compositions provided
herein, the multivalent cation can be Ca.sup.2+.
[0013] The compositions provided herein can further contain lipid
or detergent. In the compositions provided herein, the lipid or
detergent can include two or more different lipids or detergent. In
the compositions provided herein, the lipid can be from a
biological source. In the compositions provided herein, the lipid
can be rabbit brain cephalin.
[0014] The compositions provided herein can include a plasma
fraction. In the compositions provided herein, Factor V compound
can be a component of a plasma fraction. In the compositions
provided herein, the plasma fraction can be characterized by a
property selected from the group consisting of: (i) it does not
clot by itself for at least 24 hours at 37.degree. C.; (ii) it
forms a firm clot in the presence of added thrombin; (iii) it
contains at least 25% of Factor V compound that is present per unit
volume in normal human plasma; (iv) it provides a linear
anticoagulant curve using a standard anticoagulant preparation; and
(v) any combination of (i), (ii), (iii) or (iv). The compositions
provided herein can further comprise a plasma fraction
substantially free of Factors II, VII, IX, and X.
[0015] The compositions provided herein can contain additional
components, such as components of a blood clotting reaction. The
compositions provided herein can further comprise fibrinogen. The
compositions provided herein can further comprise a plasma fraction
containing fibrinogen. The compositions provided herein can further
comprise a plasma fraction containing Factor V compound, and
further can contain fibrinogen and lipid or detergent added
thereto. The compositions provided herein can further contain
prothrombin complex. The compositions provided herein can further
contain a plasma fraction containing prothrombin complex. The
compositions provided herein can further contain an anticoagulant.
The compositions provided herein can further contain a plasma
fraction containing anticoagulant. The compositions provided herein
can further contain normal plasma. The compositions provided herein
can further contain a plasma fraction with normal plasma added
thereto. The compositions provided herein can further contain
antithrombin III. The compositions provided herein can further
contain a plasma fraction containing antithrombin III. The
compositions provided herein can further contain a plasma fraction
containing Factor V compound, and can further contain prothrombin
complex, anticoagulant, lipid or detergent, and normal plasma added
thereto. The compositions provided herein can further contain a
procoagulant phospholipid. The compositions provided herein can
further contain a brain phospholipid. The compositions provided
herein can further contain an anionic phospholipid. The
compositions provided herein can further contain a cationic
detergent. The compositions provided herein can further contain a
phospholipid selected from the group consisting of
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
phosphatidylcholine, and cardiolipin. The compositions provided
herein can further contain a plurality of phospholipids. In the
compositions provided herein, the plurality of phospholipids can
contain two or more phospholipids selected from the group
consisting of phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidylcholine, and cardiolipin. The
compositions provided herein can further contain a calcium salt. In
the compositions provided herein, the calcium salt can be a calcium
halide. In the compositions provided herein, the calcium salt can
be calcium chloride. The compositions provided herein can be
substantially free of any gaseous, liquid or solid H.sub.2O. The
compositions provided herein can contain solid H.sub.2O. The
compositions provided herein can be a lyophilized composition. The
compositions provided herein can further contain a dessicant. In
the compositions provided herein, the Factor X compound can be
Factor X.sub.a. In the compositions provided herein, the Factor V
compound can be Factor V. The compositions provided herein can
further contain benzamidine. The compositions provided herein can
provide a linear correlation between clotting time and heparin
concentration range, wherein the heparin concentration range is
selected from the group consisting of 0 units/mL to about 20
units/mL, 0 units/mL to about 15 units/mL, 0 units/mL to about 12
units/mL, 0 units/mL to about 10 units/mL, 0 units/mL to about 7.5
units/mL, and 0 units/mL to about 5 units/mL. The compositions
provided herein can provide a linear correlation between clotting
time and hirudin concentration range, wherein the hirudin
concentration range is selected from the group consisting of 0
.mu.g/mL to about 20 .mu.g/mL, 0 .mu.g/mL to about 15 .mu.g/mL, 0
.mu.g/mL to about 12 .mu.g/mL, 0 .mu.g/mL to about 10 .mu.g/mL, 0
.mu.g/mL to about 7.5 .mu.g/mL, and 0 .mu.g/mL to about 5 .mu.g/mL.
The compositions provided herein can provide a linear correlation
between clotting time and heparin concentration range, wherein the
heparin concentration range is selected from the group consisting
of 0 units/mL to 20 units/mL, 0 units/mL to 15 units/mL, 0 units/mL
to 12 units/mL, 0 units/mL to 10 units/mL, 0 units/mL to 7.5
units/mL, and 0 units/mL to 5 units/mL. The compositions provided
herein can provide a linear correlation between clotting time and
hirudin concentration range, wherein the hirudin concentration
range is selected from the group consisting of 0 .mu.g/mL to 20
.mu.g/mL, 0 .mu.g/mL to 15 .mu.g/mL, 0 .mu.g/mL to 12 .mu.g/mL, 0
.mu.g/mL to 10 .mu.g/mL, 0 .mu.g/mL to 7.5 .mu.g/mL, and 0 .mu.g/mL
to 5 .mu.g/mL.
[0016] The coagulation assay compositions provided herein also can
be characterized such that Factor X compound and Factor V compound
are substantially not admixed. In another embodiment, the
coagulation assay compositions are characterized such that Factor X
compound is present in a discrete solid form and Factor V compound
is present in a discrete solid form, and wherein the discrete solid
form of Factor X compound contacts substantially only the surface
of the discrete solid form of Factor V compound. In another
embodiment, the coagulation assay compositions are characterized
such that Factor X compound is present in a discrete solid form and
Factor V compound is present in a discrete solid form, and wherein
the discrete solid form of Factor X compound contacts no more than
substantially only the surface of the discrete solid form of Factor
V compound. In another embodiment, the coagulation assay
compositions are characterized such that Factor X compound and
Factor V compound are present in discrete separate portions,
wherein the discrete separate portions contact substantially only
the surface of one another. In another embodiment, the coagulation
assay compositions are characterized such that Factor X compound
and Factor V compound are present in discrete separate portions,
wherein the discrete separate portions contact nor more than
substantially only the surface of one another. In another
embodiment, the coagulation assay compositions are characterized
such that Factor X compound is admixed with at least one admixing
compound, wherein the admixing compound is substantially not
admixed with Factor V compound. In another embodiment, the
coagulation assay compositions are characterized such that Factor X
compound is admixed with at least one admixing compound, wherein
the admixing compound is substantially unreacted with Factor V
compound. In another embodiment, the coagulation assay compositions
are characterized such that Factor V compound is admixed with at
least one admixing compound, wherein the admixing compound is
substantially not admixed with Factor X compound. In another
embodiment, the coagulation assay compositions are characterized
such that Factor V compound is admixed with at least one admixing
compound, wherein the admixing compound is substantially unreacted
with Factor X compound. In another embodiment, the coagulation
assay compositions are characterized such that Factor X compound is
not in fluid mixture with Factor V compound. In another embodiment,
the coagulation assay compositions are characterized such that
Factor X compound is not in a fluid composition containing Factor V
compound. In another embodiment, the coagulation assay compositions
are characterized such that wherein addition of an aqueous liquid
to the composition causes Factor X compound and Factor V compound
to become substantially admixed. In another embodiment, the
coagulation assay compositions are characterized such that Factor X
compound is present in excess relative to the amount of sample to
be added to the composition. In another embodiment, the coagulation
assay compositions are characterized such that Factor V compound is
present in excess relative to the amount of sample to be added to
the composition. In another embodiment, the coagulation assay
compositions are characterized such that multivalent cation salt is
present in excess relative to the amount of sample to be added to
the composition.
[0017] Also provided herein are combinations, including any of the
compositions provided herein contained in a vessel. In the
combinations provided herein, the vessel can be sealed under
vacuum. In the combinations provided herein, the vessel can be
sealed with a vapor-impermeable seal. In the combinations provided
herein, the vessel can be a glass vessel. In the combinations
provided herein, the glass vessel can be siliconized. In the
combinations provided herein, the glass vessel can be homogenously
siliconized. The combinations provided herein can further contain a
magnetically influenceable particle within the vessel. In the
combinations provided herein, the magnetically influenceable
particle can be a steel particle. In the combinations provided
herein, the steel particle can be a steel ball. In the combinations
provided herein, the steel ball can contain a diameter ranging from
about 1 mm to about 3 mm, or 1 mm to 3 mm. In the combinations
provided herein, the steel ball can contain a diameter of about 2
mm or 2 mm. In the combinations provided herein, the steel ball can
contain a diameter of about 2.4 mm or 2.4 mm.
[0018] Also provided herein are kits, where the kit can contain any
of the combinations provided herein, and one additional element.
The kits provided herein can further contain instructions for use.
In the kits provided herein, the instructions for use can include a
reference for quantitating the results of a coagulation assay. For
example, a kit can contain a data sheet for interpreting the
results of the coagulation assay, such as a data sheet that
provides heparin and/or hirudin concentration for particular
clotting times. In the kits provided herein, the instructions for
use can include a calibration curve. The kits provided herein can
further contain a blood collection device. In the kits provided
herein, the blood collection device can be a vessel. In the kits
provided herein, the blood collection device can contain a
coagulation suppressing solution. In the kits provided herein, the
blood collection device contains sodium citrate. The kits provided
herein can further contain an anticoagulant. In the kits provided
herein, the anticoagulant can be selected from the group consisting
of heparin and hirudin. The kits provided herein can further
contain a vessel mixer. In the kits provided herein, the vessel
mixer can be a vortex mixer. The kits provided herein can further
contain a magnetic device for moving a magnetic particle located in
the vessel. In the kits provided herein, the magnetic device can be
a magnetic plate. In the kits provided herein, the magnetic device
can be configured to accommodate the vessel at a pre-determined
location. The kits provided herein can further contain a device for
detecting the movement of a magnetic particle located in the
vessel. In the kits provided herein, the device for detecting can
be a magnetic plate. In the kits provided herein, the device for
detecting can be configured to accommodate the vessel at a
pre-determined location. The kits provided herein can further
contain a magnetic device for moving and detecting the movement of
a magnetic particle located in the vessel. In the kits provided
herein, the device for moving and detecting can be a magnetic
plate. In the kits provided herein, the device for moving and
detecting can be configured to accommodate the vessel at a
pre-determined location. The kits provided herein can further
contain a fluid dispensing device. In the kits provided herein, the
fluid dispensing device can be selected from the group consisting
of a disposable pipette, an adjustable pipette, a fixed volume
capillary pipette. The kits provided herein can further contain
normal human plasma.
[0019] Also provided herein are methods for preparing a blood
coagulation assay composition, by placing Factor X compound into a
vessel and placing Factor V compound into the vessel. In the
methods provided herein, any liquid placed into the vessel can be
flash frozen upon placement into the vessel. In some methods
provided herein, Factor X compound and Factor V compound are not
simultaneously present in the vessel in aqueous liquid phase. In
some methods provided herein, when Factor X compound and Factor V
compound are simultaneously present in the vessel, at least one of
Factor X compound and Factor V compound is not in contact with
liquid phase H.sub.2O. In some methods provided herein, when Factor
X compound and Factor V compound are simultaneously present in the
vessel, H.sub.2O, when present in the vessel for more than an
instant, is present in substantially only solid phase after the
instant. In some methods provided herein, when Factor X compound
and Factor V compound are simultaneously present in the vessel, at
least one of Factor X compound and Factor V compound is not in
contact with liquid phase H.sub.2O. In some methods provided
herein, the liquid phase is resultant from condensation of gaseous
H.sub.2O. In some methods provided herein, Factor X compound and
Factor V compound are not simultaneously present in the vessel in
aqueous liquid phase. In some methods provided herein, when Factor
X compound and Factor V compound are simultaneously present in the
vessel, H.sub.2O, when present in the vessel for more than an
instant, is present in substantially only solid phase after the
instant. In some methods provided herein, when Factor X compound
and Factor V compound are simultaneously present in the vessel,
H.sub.2O, when present in the vessel for more than an instant, is
present in substantially only solid phase after the instant. In
some methods provided herein, Factor X compound and Factor V
compound are not simultaneously present in the vessel in aqueous
liquid phase. In some methods provided herein, when Factor X
compound and Factor V compound are simultaneously present in the
vessel, at least one of Factor X compound and Factor V compound is
not in contact with liquid phase H.sub.2O. The methods provided
herein can further include placing an additional component into the
vessel, wherein the additional component can be selected from the
group consisting of multivalent cation, lipid or detergent,
fibrinogen, prothrombin complex, anticoagulant, normal plasma, and
combinations thereof. In some methods provided herein,
substantially no Factor II present in the vessel is converted to
Factor II.sub.a. In some methods provided herein, substantially no
fibrinogen present in the vessel is converted to fibrin. Some
methods provided herein can further include placing a plasma
fraction into the vessel. In some methods provided herein, Factor V
compound is a component of a plasma fraction. Some methods provided
herein can further include placing a plasma fraction plasma
fraction substantially free of Factors II, VII, IX, X into the
vessel. In some methods provided herein, Factor X compound and a
multivalent cation are present in a single mixture that is placed
into the vessel. In some methods provided herein, Factor V compound
and lipid or detergent are present in a single mixture that is
placed into the vessel. In some methods provided herein, Factor V
compound, lipid or detergent, and fibrinogen are present in a
single mixture that is placed into the vessel. In some methods
provided herein, Factor V compound, lipid or detergent, fibrinogen,
and benzamidine are present in a single mixture that is placed into
the vessel. In some methods provided herein, Factor V compound,
lipid or detergent, anticoagulant, prothrombin complex, and normal
plasma are present in a single mixture that is placed into the
vessel. In some methods provided herein, Factor V compound, lipid
or detergent, anticoagulant, prothrombin complex, normal plasma,
and benzamidine are present in a single mixture that is placed into
the vessel. In some methods provided herein, Factor V compound,
lipid or detergent, anticoagulant, normal plasma, fibrinogen and
benzamidine are present in a single mixture that is placed into the
vessel. In some methods provided herein, Factor V compound, lipid
or detergent, anticoagulant, antithrombin-III, Factor II,
fibrinogen and benzamidine are present in a single mixture that is
placed into the vessel. In some methods provided herein, the Factor
X compound is Factor X.sub.a. In some methods provided herein, the
Factor V compound is Factor V. Also provided herein are
compositions made by any of the methods provided herein. Such
compositions can be present in combinations, such as combinations
of a composition and a vessel. Such combinations can be present in
kit form.
[0020] Also provided herein are methods of assaying coagulation of
a sample, by mixing a sample with a coagulation assay composition
and determining the time period beginning upon mixing and ending
upon clot formation. The methods of assaying provided herein can
include mixing a sample with a coagulation assay composition,
combination or kit provided herein, and determining the time period
beginning upon mixing and ending upon clot formation. The methods
of assaying provided herein can include mixing a sample with a
coagulation assay composition provided herein, and determining the
time period beginning upon mixing and ending upon clot formation.
The methods of assaying provided herein also can include adding
blood directly into a clot monitoring vessel, mixing the blood with
a coagulation assay composition, and determining the time period
beginning upon mixing and ending upon clot formation. In the assay
methods provided herein, the sample can be selected from the group
consisting of a blood sample and a plasma sample. In the assay
methods provided herein, the amount of a coagulation inhibitor in
the sample can be determined. In the assay methods provided herein,
the sample can be blood collected from a subject, and the amount of
coagulation inhibitor in the subject's blood can be determined. In
the assay methods provided herein, the time period can correlate
linearly with the concentration of anticoagulant in the sample. In
the assay methods provided herein, the time period can correlate
linearly with a concentration range of heparin in the sample,
wherein the heparin concentration range is selected from the group
consisting of 0 units/mL to about 20 units/mL, 0 units/mL to about
15 units/mL, 0 units/mL to about 12 units/mL, 0 units/mL to about
10 units/mL, 0 units/mL to about 7.5 units/mL, and 0 units/mL to
about 5 units/mL. In the assay methods provided herein, the time
period can correlate linearly with a concentration range of hirudin
in the sample, wherein the hirudin concentration range is selected
from the group consisting of 0 .mu.g/mL to about 20 .mu.g/mL, 0
.mu.g/mL to about 15 .mu.g/mL, 0 .mu.g/mL to about 12 .mu.g/mL, 0
.mu.g/mL to about 10 .mu.g/mL, 0 .mu.g/mL to about 7.5 .mu.g/mL,
and 0 .mu.g/mL to about 5 .mu.g/mL. In the assay methods provided
herein, the time period can correlate linearly with a concentration
range of heparin in the sample, wherein the heparin concentration
range is selected from the group consisting of 0 units/mL to 20
units/mL, 0 units/mL to 15 units/mL, 0 units/mL to 12 units/mL, 0
units/mL to 10 units/mL, 0 units/mL to 7.5 units/mL, and 0 units/mL
to 5 units/mL. In the assay methods provided herein, the time
period can correlate linearly with a concentration range of hirudin
in the sample, wherein the hirudin concentration range is selected
from the group consisting of 0 .mu.g/mL to 20 .mu.g/mL, 0 .mu.g/mL
to 15 .mu.g/mL, 0 .mu.g/mL to 12 .mu.g/mL, 0 .mu.g/mL to 10
.mu.g/mL, 0 .mu.g/mL to 7.5 .mu.g/mL, and 0 .mu.g/mL to 5
.mu.g/mL.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a linear curve depicting the linear relationship
between clotting time and heparin concentration in normal human
whole blood.
[0022] FIG. 2 is a diagram depicting exemplary components of
coagulation assay compositions and steps for forming such
compositions. FIG. 2A demonstrates formation of a Factor V
compound-containing solution. FIG. 2B demonstrates combining Factor
X compound-containing solution with Factor V compound-containing
solution.
[0023] FIG. 3 is a diagram depicting exemplary components of
coagulation assay compositions for point-of-care use, and steps for
forming such compositions in accordance with the methods provided
herein.
[0024] FIG. 4 is a diagram depicting exemplary components of
coagulation assay compositions for dose-response use, and steps for
forming such compositions in accordance with the methods provided
herein.
DETAILED DESCRIPTION
I. Compositions and Methods for Measuring Anticoagulants
[0025] The compositions, combinations and methods provided herein
can be used to detect inhibitors of blood clot formation, such as
anticoagulants in the blood, and also can be used to detect other
blood components related to blood clotting, including blood
components that promote or cause blood clotting. The compositions,
combinations and methods provided herein also can be used to
provide results that accurately reflect the quantity of
anticoagulants in the blood. For example, the compositions,
combinations and methods provided herein also can be used to detect
compounds with anti-Factor X.sub.a and/or anti Factor II.sub.a
activity, such as heparin.
[0026] Provided herein are blood coagulation assay compositions
containing all components for performing a blood coagulation assay.
The compositions can be used to perform simple, short, one-step
blood coagulation assays. Blood coagulation assay compositions
provided herein contain a Factor X compound such as Factor X or
Factor X.sub.a, and also contain a Factor V compound such as Factor
V or Factor V.sub.a. Some compositions provided herein can further
contain additional components such as multivalent cation, lipid or
detergent, plasma, fibrinogen, a plasma fraction containing
fibrinogen, including a plasma fraction containing fibrinogen and
Factor V compound, and combinations thereof. Plasma fractions can
include a plasma fraction substantially free of Factors II, VII,
IX, and X. Some blood coagulation assay compositions provided
herein can further contain Factor II, prothrombin complex, or
antithrombin III. The multivalent metal can be, for example calcium
chloride or magnesium chloride, and lipid or detergent can be
either from a natural source or synthetic. Also provided herein are
combinations and kits that can contain, in addition to the blood
coagulation assay composition, an anticoagulant, a blood collection
device, a vessel for performing the assay, a mixing device, and a
coagulation monitoring device.
[0027] Also provided herein are methods for preparing blood
coagulation assay compositions, where two or more of the components
of the assay are combined in such a way that they do not
substantially react with one another. For example, the components
can be combined in a vessel such that not all components are
simultaneously present in the vessel in aqueous liquid phase.
Methods for assaying coagulation of blood are also included, where
the methods include adding blood or a blood sample to a vessel
containing a coagulation assay composition, and determining the
time period beginning upon addition of the blood or blood sample to
the composition, and terminating at an endpoint such as clot
formation. The determined time period can be used to accurately
estimate the amount of anticoagulant in the sample. For example,
the time period can be a direct function of the amount of
anticoagulant in the sample.
II. Definitions
[0028] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the invention(s) belong. All patents,
patent applications, published applications and publications,
websites and other published materials referred to throughout the
entire disclosure herein, unless noted otherwise, are incorporated
by reference in their entirety. In the event that there is a
plurality of definitions for terms herein, those in this section
prevail. Where reference is made to a URL or other such identifier
or address, it is understood that such identifiers can change and
particular information on the internet can come and go, but
equivalent information is known and can be readily accessed, such
as by searching the internet and/or appropriate databases.
Reference thereto evidences the availability and public
dissemination of such information.
[0029] As used herein, anticoagulant refers to a compound that
suppresses or inhibits coagulation or clot formation. Exemplary
anticoagulants include compounds with anti-Factor X.sub.a and/or
anti Factor II.sub.a activity, such as heparin or hirudin.
[0030] As used herein, heparin includes heparin molecules of a wide
range of molecular weights, including low molecular weight
heparins.
[0031] As used herein, a sample refers to a biological fluid from a
subject that contains one or more compounds that influence blood
coagulation. Exemplary samples include, but are not limited to
blood, plasma, serum, interstitial fluid, and lymph.
[0032] A subject can include any organism containing clotting
factors, including avian, reptilian, amphibian, mammalian such as
canine, feline, bovine, equine, porcine, rodent, ovine, caprine,
and primate, where an exemplary primate is human.
[0033] As used herein, plasma refers to the acellular fluid in
which blood cells are suspended. Plasma can be from any of a
variety of biological subjects, where exemplary plasma is from a
mammal such as canine, feline, bovine, equine, porcine, rodent,
ovine, caprine or primate. Exemplary plasma sources are bovine and
human.
[0034] As used herein, a Factor X compound refers to a compound
that has Factor X.sub.a activity or can be activated to have Factor
X.sub.a activity. Activation can take place by any known process,
including proteolysis. An exemplary compound that has Factor
X.sub.a activity is Factor X.sub.a. An exemplary Factor X compound
that can be activated to have Factor X.sub.a activity is Factor
X.
[0035] As used herein, a Factor V compound refers to a compound
that has Factor V.sub.a activity or can be activated to have Factor
V.sub.a activity. Activation can take place by any known process,
including proteolysis. An exemplary compound that has Factor
V.sub.a activity is Factor V.sub.a. An exemplary Factor V compound
that can be activated to have Factor V.sub.a activity is Factor
V.
[0036] As used herein, substantially, when used as a modifier of a
term, refers to a state in which the functional properties of the
term are not influenced beyond normal tolerance permitted by one
skilled in the art. Thus, a first compound or composition that is
substantially free of a second compound or composition refers to a
first compound or composition whose functional properties are not
influenced by the second compound or composition beyond normal
tolerance permitted by one skilled in the art. For example, a
composition that is substantially free of a blood clotting factor
such as Factors II, VII, IX or X, refers to a composition having no
such identified blood clotting factor, or so low an amount of such
identified blood clotting factor as to not influence the functional
properties of the composition beyond normal tolerance permitted by
one skilled in the art. Typically, a first compound or composition
that is substantially free of a second compound or composition
refers to a first compound or composition that contains no more
than about 25%, no more than about 20%, no more than about 10%, no
more than about 5%, no more than about 3%, no more than about 2%,
no more than about 1%, no more than about 0.5%, no more than about
0.3%, no more than about 0.2% or no more than about 0.1% of the
second compound or composition. In another example, a first
compound or composition that is substantially free of a second
compound or composition refers to a first compound or composition
that contains no more than 25%, no more than 20%, no more than 10%,
no more than 5%, no more than 3%, no more than 2%, no more than 1%,
no more than 0.5%, no more than 0.3%, no more than 0.2% or no more
than 0.1% of the second compound or composition. Similarly, a
vessel that contains substantially none of a particular compound or
composition is a vessel whose one or more components are
substantially free of the particular compound or composition.
Similarly, a compound or composition that is substantially
unreacted refers to a compound or composition whose functional
properties are not changed, relative to a completely unreacted
compound or composition, beyond normal tolerance permitted by one
skilled in the art. For example, a composition that is
substantially unreacted, refers to a composition in which the
components are completely unreacted, or the components have reacted
at so low of an amount that the functional properties of the
composition are not influenced beyond normal tolerance permitted by
one skilled in the art. Typically, a compound or composition that
is substantially unreacted refers to a compound or composition in
which no more than about 25%, no more than about 20%, no more than
about 10%, no more than about 5%, no more than about 3%, no more
than about 2%, no more than about 1%, no more than about 0.5%, no
more than about 0.3%, no more than about 0.2% or no more than about
0.1% of the compound or composition is reacted. In another example,
a compound or composition that is substantially unreacted refers to
a compound or composition in which no more than 25%, no more than
20%, no more than 10%, no more than 5%, no more than 3%, no more
than 2%, no more than 1%, no more than 0.5%, no more than 0.3%, no
more than 0.2% or no more than 0.1% of the compound or composition
is reacted. In instances where the amount of unreacted compound or
composition is difficult to measure, a substantially unreacted
compound or composition can be one in which one or more properties
of the compound or composition are at least about 80%, at least
about 90%, at least about 95%, at least about 98%, or at least
about 99% the same as completely unreacted compound or composition,
or at least 80%, at least 90%, at least 95%, at least 98%, or at
least 99% the same as completely unreacted compound or composition.
Exemplary properties include clotting time for a particular sample,
for example, normal human plasma; slope of plot of clotting time
versus anticoagulant concentration in, for example, a calibration
curve; and range of anticoagulant concentration, such as heparin
concentration, for which clotting time and anticoagulant
concentration have a linear relationship. Similarly, a compound or
composition in which substantially all of the compound or
composition has a particular property refers to a compound or
composition whose functional properties are not influenced by an
amount of compound or composition that does not have the particular
property, beyond normal tolerance permitted by one skilled in the
art. For example, a composition that is substantially aqueously
soluble refers to a compound or composition that is completely
aqueously soluble, or a compound or composition in which so low an
amount is not aqueously soluble that the functional properties of
the composition are not changed beyond normal tolerance permitted
by one skilled in the art. Typically, a compound or composition in
which substantially all of the compound or composition has a
particular property refers to a compound or composition in which no
more than about 25%, no more than about 20%, no more than about
10%, no more than about 5%, no more than about 3%, no more than
about 2%, no more than about 1%, no more than about 0.5%, no more
than about 0.3%, no more than about 0.2% or no more than about 0.1%
of the compound or composition does not have that property, or no
more than 25%, no more than 20%, no more than 10%, no more than 5%,
no more than 3%, no more than 2%, no more than 1%, no more than
0.5%, no more than 0.3%, no more than 0.2% or no more than 0.1% of
the compound or composition does not have that property.
[0037] As used herein, composition refers to a substance containing
two or more components. The term composition used herein does not
require that the two or more components be admixed.
[0038] For clarity of disclosure, and not by way of limitation, the
detailed description is divided into the subsections that
follow.
III. Components of the Compositions
[0039] The compositions provided herein can be used in assaying
coagulation of blood. The compositions provided herein contain a
Factor X compound such as Factor X or Factor X.sub.a, and also
contain a Factor V compound such as Factor V or Factor V.sub.a. The
compositions also can include a variety of additional components,
including a multivalent cation, lipid or detergent, fibrinogen,
antithrombin III, and plasma or a plasma fraction. In embodiments
in which one component of the composition contains multiple
substances, such as embodiments in which one component of the
composition is plasma or a plasma fraction containing multiple
substances, one or more of: Factor X or Factor X.sub.a, Factor V or
Factor V.sub.a, multivalent cation, lipid or detergent, fibrinogen,
antithrombin III, or other intended component, can be one of the
substances included in the plasma or plasma fraction. In such
embodiments, inclusion of plasma or the particular plasma fraction
can sufficiently contribute one of the specified components to the
composition, and separate addition of such a component, is
optional, according to the intended concentration of components of
the composition. For example, when the composition contains a
plasma fraction that includes a Factor V compound, separate
addition of a Factor V compound to the composition is not required,
but can be accomplished if desired. Thus, a variety of sources of
the components are possible in constructing the compositions
provided herein. One skilled in the art can determine according to
the teachings provided herein and the general knowledge in the art
any of a variety of different mixtures that can be used to arrive
that the compositions provided herein.
A. Factor X Compound
[0040] The compositions provided herein can contain a Factor X
compound, such as Factor X or Factor X.sub.a. Factor X.sub.a is an
activated form of Factor X. Factor X can be activated by any of a
variety of agents known in the art, including, but not limited to
Russels viper venom, thromboplastin, and citrate. In one
embodiment, the compositions provided herein can contain Factor X.
In another embodiment, the compositions provided herein can contain
Factor X.sub.a. Composition embodiments that contain Factor X can
also contain one or more Factor X-activating agents, such as
Russels viper venom. Factor X compounds can be obtained from any of
a variety of sources, as known in the art. For example Factor X
compounds can be prepared from plasma, such as bovine plasma, as
known in the art and exemplified in Yin et al., J. Biol. Chem.
243:112 (1968). Factor X compounds also can be obtained in
partially or substantially purified form from a variety of
commercial sources. For example, substantially pure Factor X.sub.a
in lyophilized form is available from Heptest Laboratories (St.
Louis, Mo.).
[0041] 1. Factor X Compound in a Composition
[0042] Factor X compound can be provided in solid form, such as
lyophilized form, or can be provided in solution, for example, in
an aqueous solution. Factor X compound can be present in a
composition such as a fluid containing one or more additional
components. Factor X compound also can be present in a composition
such as a solid containing one or more additional components. As
contemplated herein, the Factor X compound-containing compositions
described herein as fluids also can be present in solid form (e.g.,
frozen or lyophilized form). Accordingly, one skilled in the art
will understand that reference herein to components that can be
present in a Factor X compound-containing fluid composition also
includes reference to components that can be present in a Factor X
compound-containing solid composition. In one example, Factor X
compound can be present in a composition such as a fluid that also
contains one or more buffering solution components. Exemplary
buffering solution components include buffering compound, salt, and
additional components for stabilizing components of the Factor X
compound-containing composition and for facilitating solubilization
of components of the Factor X compound-containing composition, such
as lactose, bovine serum albumin (BSA), and polyethylene
glycol.
[0043] Buffering compounds can be any of a variety of compounds
known in the art for stabilizing pH in a range suitable for Factor
X or Factor X.sub.a, including amine, imine, phosphate, and sulfate
based buffering compounds, such as tris-maleate or tris-HCl. Ranges
of pH suitable for Factor X compound-containing solutions are
approximately 5 to 8, typically 7.0 to 8.0. An exemplary buffering
compound is Tris hydroxymethyl aminomethane maleate at pH 7.5.
Buffering compounds can be present at any concentration that
provides the desirable level of pH buffering, such as 5 mM-500 mM.
An exemplary concentration for buffering compound is 100 mM.
[0044] Salts can be any of a variety of compounds known in the art
as suitable in solution with Factor X or Factor X.sub.a, including,
but not limited to NaCl, NaCitrate, CaCl.sub.2 and MgCl.sub.2. Any
of a variety of salts can be present, including a combination of
salts containing monovalent ions and salts containing multivalent
ions such as divalent ions. Ranges of salt concentrations for
Factor X.sub.a solutions are approximately 10 mM to 200 mM,
typically 50 mM to 200 mM. An exemplary salt compound and
concentration is NaCl at 150 mM. Multivalent salts such as divalent
salts can be present in addition to salts such as NaCl. Typically a
multivalent salt contains a multivalent cation, such as a divalent
cation, including Ca.sup.2+ and Mg.sup.2+. An exemplary multivalent
salt compound and concentration is CaCl.sub.2 at 25 to 50 mM,
typically about 25 mM or 50 mM, or 25 mM or 50 mM.
[0045] A composition containing Factor X compound can also contain
one or more stabilizers, as known in the art. Stabilizers can aid
in preserving the activity of components such as Factor X or Factor
X.sub.a in the composition. While not intending to be limited by
the following, stabilizers can act by preventing proteins such as
Factor X or Factor X.sub.a from losing activity by, for example,
adsorption onto a surface such as glass or plastic. A variety of
stabilizers is known in the art, and includes, for example bovine
serum albumin (BSA) and polyethylene glycol (PEG). Exemplary
concentrations of stabilizers include BSA ranging from about 0.5%
to about 1.0%, typically about 1%, and PEG ranging from about 0.5%
to about 1.0%, typically about 1%. Additional exemplary
concentrations of stabilizers include BSA ranging from 0.5% to
1.0%, typically 1%, and PEG ranging from 0.5% to 1.0%, typically
1%. Additional exemplary concentrations of stabilizers include BSA
ranging from about 0.5% to about 5.0%, typically about 3%, and PEG
ranging from about 0.05% to about 0.5%, typically about 0.1%.
Additional exemplary concentrations of stabilizers include BSA
ranging from 0.5% to 5.0%, typically 3%, and PEG ranging from 0.05%
to 0.5%, typically 0.1%.
[0046] A composition containing Factor X compound can also contain
one or more compounds that facilitate solubilization. When Factor X
compound is present in solvent-free form (e.g., in lyophilized
form), one or more compounds can be used that facilitate
solvent-free Factor X compound to dissolve upon contact with an
appropriate solvent such as liquid H.sub.2O or a liquid aqueous
solution. Any of a variety of solubilizing compounds known in the
art can be used, including, but not limited to, lactose. Exemplary
concentrations of solubilizing compounds include lactose ranging
from about 1.0% to about 20%, typically about 5 or about 10%.
Additional exemplary concentrations of solubilizing compounds
include lactose ranging from 1.0% to 20%, typically 5 or 10%.
[0047] In addition, glycine can be added to a composition
containing Factor X compound. Exemplary concentrations of glycine
can range from about 0.5% to about 2%, typically about 1%.
Additional exemplary concentrations of glycine can range from 0.5%
to 2%, typically 1%.
[0048] In another embodiment, a composition such as a fluid
composition can contain Factor X compound and one or more compounds
that are active in a blood clotting assay, including but not
limited to, a blood clotting enzyme, a substrate of a blood
clotting enzyme, a modulator of blood clotting enzyme activity
(e.g., an inhibitor or activator), and combinations thereof.
Exemplary compounds that can be in a composition such as a fluid
composition with Factor X compound include, but are not limited to,
multivalent cation, lipid or detergent, fibrinogen, and
combinations thereof. Additional components that can be added to a
composition containing Factor X compound include Factor II,
prothrombin complex, and combinations thereof. Factor X.sub.a is
typically not provided in fluid solution with antithrombin III.
Further, in some embodiments, Factor X compound is not provided in
fluid solution with Factor V compound.
B. Factor V Compound
[0049] The composition provided herein can contain Factor V
compound. Factor V compound can include Factor V and Factor
V.sub.a, and can be obtained by any of a variety of methods known
in the art, as exemplified in Dahlback, J Clin Invest. 66:583-91
(1980), Kane, et al., J. Biol. Chem. 256:1002-1007 (1981), Smirnov
et al., J Biol Chem 273:9031-9040 (1998), U.S. Pat. No. 4,851,336
to Yin, and U.S. Pat. Pub. No. 20030104508. For example, a Factor
V-containing plasma fraction can be prepared from plasma, such as
bovine plasma, as known in the art and exemplified in U.S. Pat. No.
4,851,336 to Yin. Factor V is also available from commercial
suppliers, including CalBiochem (San Diego, Calif.). Methods also
are known in the art for activating Factor V to Factor V.sub.a. The
methods and commercial sources can be used to obtain Factor V
compound in partially purified form or substantially purified
form.
[0050] 1. Factor V Compound Composition
[0051] Factor V compound can be provided in solid form, such as
lyophilized form, or can be provided in solution, for example, in
an aqueous solution. Factor V compound can be present in a
composition such as a fluid containing one or more additional
components. Factor V compound also can be present in a composition
such as a solid containing one or more additional components. As
contemplated herein, the Factor V compound-containing compositions
described herein as fluids also can be present in solid form (e.g.,
frozen or lyophilized form). Accordingly, one skilled in the art
will understand that reference herein to components that can be
present in a Factor V compound-containing fluid composition also
includes reference to components that can be present in a Factor X
compound-containing solid composition. In one example, Factor V
compound can be present in a composition such as a fluid
composition containing one or more buffering solution components.
In another example, Factor V compound can be present in a
biological fluid or a fraction thereof. In another example Factor V
compound can be in a composition containing one or more additional
components of a coagulation assay composition.
[0052] a. Buffer Conditions
[0053] Factor V compound can be present in a composition such as a
fluid that also contains one or more buffering solution components,
as known in the art. Exemplary buffering solution components
include buffering compound, salt, stabilizers and solubilizers, as
described herein for compositions containing Factor X compound.
[0054] Factor V compound buffer conditions can vary as desirable
for desired properties. For example, salt concentration, such as
sodium chloride concentration can be about 250 mM to about 750 mM,
such as about 500 mM. In another example, salt concentration, such
as sodium chloride concentration can be 250 mM to 750 mM, such as
500 mM. In another example, PEG can be present at about 0.1% to
about 1%, such as about 0.25%. In another example, BSA can be
present at about 1% to about 10%, such as about 4%. In another
example, PEG can be present at 0.1% to 1%, such as 0.25%. In
another example, BSA can be present at 1% to 10%, such as 4%. In
another example, compounds such as multivalent cation salt, lactose
and glycine are not added to the Factor V compound solution.
[0055] b. Factor V Compound with Other Components
[0056] Factor V compound also can be provided as one component of a
composition such as a fluid composition of substances, for example,
in a biological fluid such as plasma or a plasma fraction. In one
embodiment, such a composition can contain Factor V compound and
one or more additional blood components. In another embodiment,
Factor V compound can be in a composition containing fibrinogen, or
a composition containing antithrombin III, or a composition
containing both antithrombin III and fibrinogen. In another
embodiment, Factor V compound can be in a composition containing
lipid or detergent, normal plasma, prothrombin complex,
anticoagulant, or combinations thereof.
[0057] One example of a plasma fraction containing Factor V
compound is a plasma fraction that contains Factor V and,
optionally, fibrinogen, and is substantially free of Factors II,
VII, IX and X. Methods for preparing such a plasma fraction are
known in the art, as exemplified in U.S. Pat. No. 4,851,336. An
exemplary method for producing such a plasma fraction can be
performed by treating mammalian blood with a coagulation
suppressing composition, and then removing blood cells to produce
plasma, then treating the plasma with at least one separation step
to remove coagulation Factors II, VII, IX and X, then treating the
resulting plasma fraction with any further desired separation steps
and recovering therefrom a protein fraction containing coagulation
Factor V and optionally fibrinogen, and then treating the protein
fraction to remove therefrom soluble ammonium salt and insoluble
proteins. The resultant plasma fraction can be buffered and can
also be admixed with one or more compounds, such as fibrinogen,
lipid or detergent, prothrombin complex, anticoagulant, normal
plasma, or combinations thereof.
[0058] Each of the steps of the exemplary protocol can be performed
by any of a variety of methods known in the art, as exemplified in
U.S. Pat. No. 4,851,336. Brief descriptions of exemplary methods
for performing each step are as follows. In the blood treatment
step, blood can be treated with any of a variety of coagulation
suppressing compounds or compositions, as known in the art, for
example, a 3.8% sodium citrate solution, a sodium or potassium
oxalate solution, or EDTA. Typically, when a sodium citrate
solution is used, it is mixed with blood in ratios of about 9:1
blood:sodium citrate, or ratios of 9:1 blood:sodium citrate. The
treated blood can then be treated in one or more steps to separate
blood cells from the plasma, as known in the art. For example, the
treated blood can be centrifuged at 2500.times.g for 15 minutes.
The resultant plasma can then be used in subsequent treatment
steps.
[0059] The plasma can be treated to separate Factors II, VII, IX
and X from Factor V. Any of a variety of separation methods can be
used to perform this step, as known in the art, including, but not
limited to, liquid chromatographic methods such as affinity
chromatography, ion exchange chromatography, or gel filtration
chromatography, and precipitation methods including salt
precipitation or alcohol precipitation. In some embodiments,
fibrinogen can also be present in the fraction containing Factor V.
In an exemplary method, the plasma can be treated to have Factors
II, VII, IX and X become no longer dissolved in the plasma solution
while Factor V, and, optionally, fibrinogen, remain dissolved. Any
of a variety of precipitation methods known for removing Factors
II, VII, IX and X from the plasma solution while Factor V, and,
optionally, fibrinogen, remains dissolved, can be used. In an
exemplary method, 1 liter of plasma can have dissolved therewith
7.35 g of trisodium citrate, followed by addition of 20.825 g of
barium chloride (to achieve an overall barium chloride
concentration of about 0.1 M in the plasma mixture) at room
temperature over a period of 1-2 hours. Methods using solutions
containing citrate and barium ions as precipitants can be performed
at any of a variety of citrate and barium ion concentrations known
to remove Factors II, VII, IX and X from the plasma solution,
without also substantially removing Factor V, and, optionally,
fibrinogen, from the plasma solution, as known in the art. As an
alternate example, when the blood from the first step of plasma
isolation is treated with a coagulation suppressor such as sodium
or potassium oxalate, the plasma treatment step can include
treating the plasma with barium sulfate, which can result in
Factors II, VII, IX and X being removed from the plasma solution
while Factor V, and, optionally, fibrinogen, remain dissolved.
Methods for using barium sulfate to remove Factors II, VII, IX and
X from plasma without substantially removing Factor V, and
optionally, fibrinogen, are known in the art, as exemplified in
page 592, Human Blood Coagulation, Haemostasis and Thrombosis,
edited by Biggs, 1972. The resultant solid can then be separated
from the liquid by any of a variety of methods known in the art,
including, but not limited to, centrifugation and filtration. For
example, centrifugation of the treated plasma can be performed, for
example, at 3000.times.g for 20 minutes, where the resulting pellet
plasma fraction typically contains Factors II, VII, IX and X, and
the supernatant plasma fraction typically contains Factor V, and,
optionally, fibrinogen. The Factor V-containing supernatant plasma
fraction can then be further treated.
[0060] The supernatant plasma fraction can be treated by a further
step in order to separate Factor V, and, optionally, fibrinogen,
from other non-clotting proteins. Any of a variety of separation
methods can be used to perform this step, such as chromatographic
and precipitation methods, as provided herein or otherwise known in
the art. In one exemplary method, the supernatant plasma fraction
can be treated with a salt, such as ammonium sulfate, to
precipitate the Factor V and any other intended components such as
fibrinogen. The use of ammonium sulfate for the fractionation of
Factor V is known in the art, as exemplified in "Human Blood
Coagulation and Its Disorders" edited by Biggs & MacFarlane, p.
54, 3rd edition 1962. The use of ammonium sulfate to precipitate
fibrinogen also is known in the art, as exemplified in "The
Biochemistry of Blood Coagulation" by T. Astrup, in Acta
Physiologica Scandanavia, Supplement 21, 1944. In an exemplary
method, ammonium sulfate can be added to the supernatant plasma
fraction at a concentration of about 270 g per liter or 270 g per
liter of the plasma fraction, at room temperature, resulting in an
about 40% to 45% or 40% to 45% saturated solution. Such mixture can
be incubated at room temperature for 1-2 hours. Other salts or
other ammonium sulfate concentrations can also be used to achieve
similar results, as is known in the art. For example a saturated
ammonium sulfate-plasma fraction can contain a range from about 30%
to about 60% or 30% to 60% ammonium sulfate, typically from about
35% to about 50% or 35% to 50% ammonium sulfate. Generally in such
methods, an ammonium sulfate concentration of about 30% or 30%
saturation results in fibrinogen precipitation, and an ammonium
sulfate concentration between about 30% or 30% saturation to about
40% or 40% saturation results in Factor V precipitation. The
resultant precipitate of the mixture can be separated from the
liquid by any of a variety of methods known in the art, such as
centrifugation or filtration. For example, the mixture can be
centrifuged at 4000.times.g for 15 minutes at room temperature.
Typically for an ammonium sulfate precipitation such as that
presented in this paragraph, the fraction containing Factor V, and
optionally, fibrinogen, is the precipitate, which forms a pellet
upon centrifugation.
[0061] The pellet plasma fraction can then be resuspended in an
aqueous solution, as known in the art. The aqueous solution can be
of any volume or composition suitable for stabilizing Factor V and
other intended components of the pellet, such as, but not limited
to, fibrinogen. For example, the pellet can be resuspended in a
volume of distilled water equal to 30-40% of the original plasma
volume. The resuspended pellet plasma fraction can then be treated
to remove ammonium ion from the solution. Removal of solutes
including ions such as ammonium from a solution can be accomplished
by any of a variety of methods known in the art, typically by use
of a membrane for filtration and/or solute/solvent exchange. An
exemplary method is dialysis. For example, the resuspended pellet
plasma fraction can be dialysed against a 0.9% NaCl solution or
equivalent, including tap water. Typically, such solute removal
steps are performed by adding barium chloride to a sample of the
dialysis solution and monitoring for the precipitation of barium
sulfate. When precipitate is no longer formed, solute exchange is
sufficiently complete.
[0062] After solute exchange such as dialysis, the resuspended
pellet plasma fraction can be treated in one or more steps to have
any solids or particulate matter removed. Any of a variety of
methods known in the art for removing solids from a solution can be
used, including centrifugation or filtration. For example, the
mixture can be centrifuged at 3000.times.g for 15 minutes at room
temperature.
[0063] The plasma fraction resultant from the above procedure or
equivalents thereof known in the art, can then have added thereto
one or more buffering solution components, including, but not
limited to, buffering compounds, salts, bovine serum albumin (BSA),
lactose, and polyethylene glycol. Such buffering solution
components are further described herein elsewhere. An exemplary
buffering solution can contain 2.50 g polyethyleneglycol, 9.0 g
NaCl, 98.88 g Tris-maleate, and 100 g lactose per liter of
distilled water, at a pH of 7.5, where the buffering solution is
combined with the plasma fraction at a ratio of 9:1 plasma
fraction:buffering solution.
[0064] The buffered plasma fraction can also have added thereto
lipid or detergent such as brain phospholipids including cephalin.
The amount of added lipid or detergent can be a function of the
concentration of Factor X compound selected for the assay. For
example, the more concentrated the Factor X compound present in the
assay system, the less amount of lipid added to the Factor
V-containing composition. The resulting plasma fraction admixture
can be described in terms of one or more of its components, in
terms of one or more steps in its method of preparation, or in
terms of one or more of its properties. However, regardless of the
manner described, one skilled in the art, based on the teachings
provided herein and the knowledge of skill in the art, will
recognize that a range of related plasma fraction products can
contain the intended components and intended amounts in suitable
form for use in the methods and compositions provided herein.
[0065] The resultant plasma fraction is characterized as containing
Factor V. The Factor V-containing plasma fraction can also,
optionally be characterized by one or more additional properties,
including, but not limited to: it contains fibrinogen, it does not
clot by itself for at least 24 hours at 37.degree. C., it can form
a firm clot in the presence of added thrombin, it contains at least
25% of Factor V that is present per unit volume in normal human
plasma, and it can be used in a coagulation assay to provide a
linear anticoagulant curve using a standard anticoagulant
preparation, such as a standard heparin preparation.
[0066] 2. Optional Components
[0067] In another embodiment, a composition such as a fluid
composition can contain Factor V compound and one or more compounds
that are active in a blood clotting assay, including but not
limited to, a blood clotting enzyme, a substrate of a blood
clotting enzyme, a modulator of blood clotting enzyme activity
(e.g., an inhibitor or activator), and combinations thereof.
Exemplary compounds that can be in a composition with Factor V
compound include, but are not limited to lipid or detergent,
fibrinogen, antithrombin III, Factor II, prothrombin complex,
anticoagulant, normal plasma, and combinations thereof. For
example, Factor V compound or a plasma fraction containing Factor V
compound can further contain lipid or detergent. In another
example, a Factor V compound or a plasma fraction containing Factor
V compound can further contain fibrinogen. In another example,
Factor V compound or a plasma fraction containing Factor V compound
can further contain antithrombin III. In another example, Factor V
compound or a plasma fraction containing Factor V compound can
further contain Factor II. In another example, Factor V compound or
a plasma fraction containing Factor V compound can further contain
prothrombin complex. In another example, Factor V compound or a
plasma fraction containing Factor V compound can further contain
normal plasma. In another example, Factor V compound or a plasma
fraction containing Factor V compound can further contain
anticoagulant. In another example, Factor V compound or a plasma
fraction containing Factor V compound can further contain
combinations of two or more of lipid or detergent, fibrinogen,
antithrombin III, factor II, prothrombin complex, normal plasma,
and anticoagulant. In another example, a composition containing
Factor V compound and fibrinogen or a plasma fraction containing
Factor V compound and fibrinogen can further contain lipid or
detergent. In another example, a composition containing Factor V
compound and fibrinogen or a plasma fraction containing Factor V
compound and fibrinogen can further contain antithrombin III. In
another example, a composition containing Factor V compound and
fibrinogen or a plasma fraction containing Factor V compound and
fibrinogen can further contain Factor II or prothrombin complex. In
another example, a composition containing Factor V compound and
fibrinogen or a plasma fraction containing Factor V compound and
fibrinogen can further contain normal plasma. In another example, a
composition containing Factor V compound and fibrinogen or a plasma
fraction containing Factor V compound and fibrinogen can further
contain anticoagulant. In another example, a composition containing
Factor V compound and fibrinogen or a plasma fraction containing
Factor V compound and fibrinogen can further contain two or more of
phospholipid or detergent, antithrombin III, Factor II, prothrombin
complex, normal plasma, and anticoagulant. In one example, a
composition containing Factor V compound, such as a plasma fraction
containing Factor V compound, can further contain lipid or
detergent, normal plasma, prothrombin complex, and
anticoagulant.
C. Additional Components
[0068] The coagulation assay compositions provided herein can
include components in addition to Factor X compound and Factor V
compound. As provided herein, additional components can be
biological compositions such as normal plasma, or can be blood
clotting factors such as fibrinogen, prothrombin complex, Factor II
or antithrombin III, or other components that influence clotting
such as anticoagulant, multivalent cations, and lipid or
detergent.
[0069] Fibrinogen, prothrombin complex, Factor II, normal plasma,
anticoagulant, and antithrombin III can readily be obtained by
methods known in the art or by commercial sources.
[0070] When a multivalent cation salt and lipid or detergent are
included, they can each be present in substantially pure form, or
can be present in a composition such as a fluid composition,
including a composition containing one or more components that are
active in a blood clotting assay, including, but not limited to, a
blood clotting enzyme, a blood clotting enzyme substrate, a blood
clotting enzyme modulator, and combinations thereof. For example,
multivalent cation salt and/or lipid or detergent can be in a
composition such as a fluid composition with Factor X.sub.a, Factor
V, fibrinogen, normal plasma, prothrombin complex, anticoagulant,
antithrombin III, and combinations thereof.
[0071] In some embodiments, when multivalent cation salt and lipid
or detergent are added rapidly together in a fluid composition,
precipitate may form. Accordingly, when multivalent cation salt and
lipid or detergent are to be present in a fluid composition, they
can be present in a fluid that also contains a compound or
composition that suppresses precipitate formation. Compounds and
compositions that suppress precipitate formation in fluids
containing multivalent cation salt and lipid or detergent are known
in the art. In one example, a plasma protein, such as BSA, can
suppress precipitate formation in fluids containing multivalent
cation salt and lipid or detergent. In another example, a plasma
fraction can suppress precipitate formation in fluids containing
multivalent cation salt and lipid or detergent. In another example
a plasma fraction containing Factor V compound can suppress
precipitate formation in fluids containing multivalent cation salt
and lipid or detergent. When multivalent cation salt and lipid or
detergent are to be added to a fluid, typically a first of the two
is fully added and fully mixed prior to adding the second. In one
example, multivalent cation salt is fully mixed into a fluid
composition prior to adding lipid or detergent. An exemplary fluid
into which multivalent cation salt and lipid or detergent can be
mixed is a plasma fraction containing Factor V compound, such as a
buffered plasma fraction containing Factor V compound, as provided
herein or otherwise known in the art. Additional compounds and
compositions which can be mixed with multivalent cation salt and/or
lipid or detergent to form a composition such as a fluid
composition, include, but are not limited to antithrombin III,
Factor II, Factor X compound, fibrinogen, anticoagulant, normal
plasma and prothrombin complex.
[0072] 1. Multivalent Cation-Containing Salt
[0073] The salts used in the compositions provided herein can
include any of a variety of salts containing multivalent cations
that are known to promote coagulation. In one embodiment, the
multivalent cation is a divalent cation. Any of a variety of
divalent cations known to promote coagulation can be used,
including, but not limited to, calcium ion and magnesium ion.
Typically the counteranion of the cation can be any anion that does
not inhibit coagulation. Any of a variety of anions can be used as
the counteranion, as known in the art. Exemplary anions include
halides, carboxylates, sulfates, sulfites, phosphates, nitrates,
and nitrites. In one embodiment, divalent cation halides such as
calcium chloride and magnesium chloride are used. Any of a variety
of multivalent cation salts such as calcium salts is available from
commercial sources, such as Sigma-Aldrich (St. Louis, Mo.).
[0074] In one embodiment, compositions provided herein also include
any coagulation assay composition known in the art, including
compositions not containing both Factor X compound and Factor V
compound, or compositions not containing either Factor X compound
or Factor V compound, having added thereto a multivalent cation. A
variety of coagulation assay compositions are known in the art, and
include ACT and APTT assay compositions, as exemplified in U.S.
Pat. Nos. 6,699,718, 6,528,273 and 6,140,062. Multivalent cation,
such as divalent cation including calcium and magnesium ions, can
be present in such coagulation assay compositions, while the
components of the coagulation assay compositions are substantially
unreacted or unactivated. Multivalent cation, such as divalent
cation including calcium and magnesium ions, can be present in such
coagulation assay compositions, where the compositions are
substantially free of liquid or gaseous H.sub.2O.
[0075] 2. Lipid or Detergent
[0076] The coagulation assay compositions provided herein also can
include lipid or detergent. Typical lipid used include
phospholipids such as phosphatidylcholine, phosphatidylserine,
phosphatidylethanolamine, phosphatidylinositol, and cardiolipin.
Phospholipids used in the compositions provided herein can include
any phospholipid known to promote coagulation (i.e., any
procoagulant phospholipid), including biological isolates
containing phospholipids; for example, a typical phospholipid in
the compositions provided herein is phospholipids from rabbit brain
(e.g., cephalin). In another example, the phospholipid can be
soybean phospholipid. Any of a variety of procoagulant
phospholipids known in the art can be used, including, but not
limited to, cephalins. Any of a variety of procoagulant
phospholipids is available from commercial sources, such as
Sigma-Aldrich (St. Louis, Mo.). Procoagulant phospholipids also can
be isolated from biological sources according to any of a variety
of methods known in the art. For example, brain phospholipids such
as cephalin can be isolated from brain, as known in the art, and
exemplified in Bell et al., Nature, 174:880 (1954). Exemplary
brains for isolation of brain phospholipids such as cephalin are
rabbit brain or bovine brain.
[0077] Detergent used in the compositions provided herein can
include any detergent known to promote coagulation. Typical
detergents used include cationic detergents such as CTAB
(cetyltrimethyl-ammonium bromide). Exemplary detergents include,
but are not limited to, CTAB and phosphatidic acid. Any of a
variety of detergents is available from commercial sources, such as
Sigma (St. Louis, Mo.).
[0078] Also encompassed within the use of lipid or detergent in the
compositions provided herein are compositions containing two or
more different lipids or detergents, which can include two or more
different lipids, two or more different detergents, or combinations
of at least one lipid and at least one detergent. Compositions
containing two or more different lipids or detergents can be formed
by combining two pure or substantially pure lipids or detergents,
or can be biological isolates containing two or more lipids or
detergents, such as a lipid isolate from a biological source
containing two or more lipids or detergents; an exemplary
biological isolate is a lipid isolate from brain, such as brain
cephalin. A variety of mixtures of lipids, detergents, and lipids
and detergents, are known in the art to have procoagulant activity.
For example, phophatidylserine can be used in combination with
CTAB. One skilled in the art will readily understand any of a
variety of mixtures that can be used, as is well known and
exemplified in publications such as Barton et al., J. Lipid Res.
11:87-95 (1970) and Papahadjoupolus et al., Proc. Soc. Exp. Biol.
Med. 111:412 (1962).
D. Optional Components
[0079] The coagulation assay compositions provided herein can also
contain a variety of additional components. Exemplary components
are those that contain one or more compounds that are active in a
blood clotting assay, including, but not limited to, a blood
clotting enzyme, a blood clotting enzyme substrate, or a blood
clotting enzyme modulator. Such components include, but are not
limited to, normal plasma, prothrombin complex, Factor II,
anticoagulant, and antithrombin III. As described elsewhere herein,
any such additional component can be added with any of Factor X
compound, Factor V compound, multivalent cation salt, or lipid or
detergent, or with any composition containing Factor X compound,
Factor V compound, multivalent cation salt, or lipid or detergent,
such as a plasma fraction containing Factor X compound, Factor V
compound, multivalent cation salt, or lipid or detergent. However,
typically antithrombin III is not in fluid mixture with Factor
X.sub.a. In some embodiments, Factor X compound is not in fluid
mixture with Factor V compound or a fluid composition containing
Factor V compound.
E. Components when Combined
[0080] The components described herein can be combined to form the
coagulation assay composition provided herein. Methods for
combining the components are provided herein elsewhere. Typically,
the coagulation assay compositions, when all components are
combined, will contain substantially no H.sub.2O in liquid or
gaseous form prior to use in performing a coagulation assay. While
not intended to be limited to the following, when all components
are present in a composition, and the composition contains a
significant amount of H.sub.2O in liquid form (including condensate
from H.sub.2O vapor), the components may react, degrade, or
otherwise be modified to render the composition ineffective or
undesirably less effective in coagulation assays. The compositions
provided herein can include H.sub.2O in solid form, provided that,
prior to use in performing the coagulation assay, the ambient
conditions are not changed to a point in which H.sub.2O is in
contact with the composition in liquid form, for example the
ambient temperature of the composition is not raised to a
temperature at which a substantial amount of H.sub.2O transitions
to liquid or gaseous form.
[0081] In one embodiment, the coagulation assay compositions, when
all components are combined, can also possess one or more
additional characteristics. In one characteristic, the components
of the composition can be present in substantially unreacted form.
For example, components of the composition can be substantially
unreacted with one another. In another characteristic, the
components of the composition can be present in substantially
unactivated form. For example, components of the composition can be
in a state where any coagulation process is substantially
unactivated. In another example, Factor X compound in the
composition is substantially unbound by antithrombin III. In
another characteristic, the composition is substantially free of
products of a clotting reaction. For example, the composition can
be substantially free of Factor II.sub.a and/or can be
substantially free of fibrin. In another characteristic,
substantially all of the Factor Xa of the composition is aqueously
soluble. In another characteristic, substantially all of the
calcium salt of the composition is aqueously soluble.
[0082] In another embodiment, the coagulation assay composition is
characterized such that Factor X compound and Factor V compound are
substantially not admixed. For example, Factor X compound can be in
a solid form, and Factor V can be in solid form, where the Factor X
compound solid can be attached to or in contact with the Factor V
compound solid, but these solids are not substantially admixed.
[0083] In another embodiment, the coagulation assay composition is
characterized such that Factor X compound is present in a discrete
solid form and Factor V compound is present in a discrete solid
form, and wherein the discrete solid form of Factor X compound
contacts substantially only the surface of the discrete solid form
of Factor V compound. In another embodiment, the coagulation assay
composition is characterized such that Factor X compound is present
in a discrete solid form and Factor V compound is present in a
discrete solid form, and wherein the discrete solid form of Factor
X compound contacts no more than substantially only the surface of
the discrete solid form of Factor V compound. In another
embodiment, the coagulation assay composition is characterized such
that Factor X compound and Factor V compound are present in
discrete separate portions, wherein the discrete separate portions
contact substantially only the surface of one another. In another
embodiment, the coagulation assay composition is characterized such
that Factor X compound and Factor V compound are present in
discrete separate portions, wherein the discrete separate portions
contact nor more than substantially only the surface of one
another. In another embodiment, the coagulation assay composition
is characterized such that Factor X compound is admixed with at
least one admixing compound, wherein the admixing compound is
substantially not admixed with Factor V compound. In another
embodiment, the coagulation assay composition is characterized such
that Factor X compound is admixed with at least one admixing
compound, wherein the admixing compound is substantially unreacted
with Factor V compound. In another embodiment, the coagulation
assay composition is characterized such that Factor V compound is
admixed with at least one admixing compound, wherein the admixing
compound is substantially not admixed with Factor X compound. In
another embodiment, the coagulation assay composition is
characterized such that Factor V compound is admixed with at least
one admixing compound, wherein the admixing compound is
substantially unreacted with Factor X compound. In another
embodiment, the coagulation assay composition is characterized such
that Factor X compound is not in fluid mixture with Factor V
compound. In another embodiment, the coagulation assay composition
is characterized such that Factor X compound is not in a fluid
composition containing Factor V compound. In another embodiment,
the coagulation assay composition is characterized such that
wherein addition of an aqueous liquid to the composition causes
Factor X compound and Factor V compound to become substantially
admixed. In another embodiment, the coagulation assay composition
is characterized such that Factor X compound is present in excess
relative to the amount of sample to be added to the composition. In
another embodiment, the coagulation assay composition is
characterized such that Factor V compound is present in excess
relative to the amount of sample to be added to the composition. In
another embodiment, the coagulation assay composition is
characterized such that multivalent cation salt is present in
excess relative to the amount of sample to be added to the
composition.
[0084] In another characteristic, the composition can have reactive
properties, where, upon addition of blood or a blood sample to the
composition, a clotting reaction initiates. In another
characteristic, the composition can have reactive properties, where
a measured clotting time can be used to determine the amount of
anticoagulant, such as heparin, in the sample. In another
characteristic, the composition can have reactive properties, where
the amount of anticoagulant, such as heparin, in a sample
containing anticoagulant amounts ranging from 0 units/mL to about
15 units/mL or from 0 units/mL to 15 units/mL, is linearly
correlated with clotting time. In another characteristic, the
composition can have reactive properties, where the amount of
anticoagulant, such as heparin, in a sample containing
anticoagulant amounts ranging from 0 units/mL to about 10 units/mL
or 0 units/mL to 10 units/mL, is linearly correlated with clotting
time. In another characteristic, the composition can have reactive
properties, where the amount of anticoagulant, such as heparin, in
a sample containing anticoagulant amounts ranging from 0 units/mL
to about 7.5 units/mL or 0 units/mL to 7.5 units/mL, is linearly
correlated with clotting time. In another characteristic, the
composition can have reactive properties, where, upon addition of a
normal blood sample or a normal plasma sample to the composition, a
clotting reaction terminates within 60 seconds of addition of the
sample. In another characteristic, the composition can have
reactive properties, where, upon addition of a normal blood sample
or a normal plasma to the composition, a clotting reaction
terminates within 120 seconds of addition of the sample. In another
characteristic, the composition can have reactive properties,
where, upon addition of a normal blood sample or a normal plasma
sample to the composition, a clotting reaction terminates within
300 seconds of addition of the sample. In another characteristic,
the composition can have reactive properties, where, upon addition
of a normal blood sample or a normal plasma to the composition, a
clotting reaction terminates within 600 seconds of addition of the
sample. In another embodiment, the composition can have reactive
properties, wherein, upon addition of a normal plasma or blood
sample, clotting time ranges from about 10 to about 13 seconds or
10 to 13 seconds, and, upon addition of a normal plasma or blood
sample containing 3 units/mL of heparin, clotting time ranges from
about 35 to about 50 seconds or 35 to 50 seconds. In another
embodiment, the composition can have reactive properties, wherein,
upon addition of a normal plasma or blood sample, clotting time
ranges from about 15 to about 20 seconds or 15 to 20 seconds, and,
upon addition of a normal plasma or blood sample containing 3
units/mL of heparin, clotting time ranges from about 40 to about 55
seconds or 40 to 55 seconds. In another embodiment, the composition
can have reactive properties, wherein, upon addition of a normal
plasma or blood sample, clotting time ranges from about 13 to about
17 seconds or 13 to 17 seconds, and, upon addition of a normal
plasma or blood sample containing 7.5 units/mL of heparin, clotting
time is less than about 100 seconds or 100 seconds. Any of a
variety of additional combinations of components can be prepared to
have related clotting time properties, according to the teachings
provided herein and the desired properties of the composition.
[0085] In another characteristic, the assay composition can
accurately determine the activity and/or concentration of
anti-Factor X.sub.a and/or anti Factor II.sub.a compounds
regardless of the presence of compounds such as aprotinin in a
sample. Aprotinin can be administered to a subject to reduce the
incidence of post-operative abnormal bleeding and blood transfusion
due to activation of the fibrinolytic pathway. However, aprotinin
in a subject can interfere with the results of some types of
coagulation assays of samples from the subject. The present
coagulation assay composition can be performed to accurately
determine the activity and/or concentration of anti-Factor X.sub.a
and/or anti Factor II.sub.a compounds such as heparin or hirudin,
without having the accuracy or precision influenced by the presence
of aprotinin in the sample.
[0086] 1. General Combination Guidelines
[0087] In forming the coagulation assay composition provided
herein, the components can be combined in relative amounts
according to the desired clotting properties resultant from
combining the components. In one embodiment, component ratios are
determined according to the desired clotting time of the
coagulation assay composition. For example, a Factor X compound
composition can be combined with a Factor V compound composition
and with blood or plasma, and the clotting time can be measured.
One or more components can be increased or decreased in relative
amount in achieving the desired coagulation properties. In one
embodiment, the composition is formed such that Factor X compound
is in excess (e.g., is not a rate limiting component). In another
embodiment, the composition is formed such that Factor V compound
is in excess (e.g., is not a rate limiting component). In another
embodiment, the composition is formed such that Factor X compound
and Factor V compound are in excess (e.g., are not rate limiting
components). In another embodiment, the composition is formed such
that multivalent cation, including divalent cation such as calcium
or magnesium ion is in excess (e.g., is not a rate limiting
component). In another embodiment, the composition is formed such
that Factor X compound, Factor V compound and multivalent cation
are in excess (e.g., are not rate limiting components). Typically,
referral to excess of a component indicates that the amount of the
component is sufficiently greater than the amount of at least one
other factor involved in the coagulation cascade such that, for the
amount of sample to be used, the rate of coagulation is not
influenced by small changes in the amount of the component.
[0088] Coagulation properties typically include clotting time,
where the clotting time for the resultant composition can be any
amount of time suitable for the corresponding use that also
provides sufficiently accurate results. For purposes of the present
description, clotting time represents the time required for the
coagulation assay composition to form a solid clot after addition
of normal human blood (unless otherwise indicated) with the
coagulation assay composition. Clotting times can be any of a wide
range of times, from as short as about 1 second, to as long as
multiple hours. Typically, clotting times will range from about 5
seconds to about 600 seconds or 5 seconds to 600 seconds, from
about 5 seconds to about 300 seconds or 5 seconds to 300 seconds,
from about 5 seconds to about 120 seconds or 5 seconds to 120
seconds, or from about 5 seconds to about 90 seconds or 5 seconds
to 90 seconds. Exemplary clotting times can range from about 10
seconds to about 45 seconds or 10 seconds to 45 seconds.
[0089] Additional coagulation properties of the coagulation assay
composition can include the ability for the clotting time and
anticoagulant concentration to be linearly correlated. For example,
when the anticoagulant is heparin, the clotting time resultant from
combining the coagulation assay composition with normal human blood
containing any of a range of heparin concentrations will form a
straight line when clotting time is plotted against heparin
concentration. Typically, the heparin concentrations of the range
are biologically relevant heparin concentrations. The compositions
provided herein can provide a linear correlation between clotting
time and a variety of heparin concentrations, including 0 units/mL
to about 20 units/mL, 0 units/mL to about 15 units/mL, 0 units/mL
to about 12 units/mL, 0 units/mL to about 10 units/mL, 0 units/mL
to about 7.5 units/mL, or 0 units/mL to about 5 units/mL. The
compositions provided herein can provide a linear correlation
between clotting time and a variety of heparin concentrations,
including 0 units/mL to 20 units/mL, 0 units/mL to 15 units/mL, 0
units/mL to 12 units/mL, 0 units/mL to 10 units/mL, 0 units/mL to
7.5 units/mL, or 0 units/mL to 5 units/mL. In another example, when
the anticoagulant is hirudin, the clotting time resultant from
combining the coagulation assay composition with normal human
plasma containing any of a range of hirudin concentrations will
form a straight line when clotting time is plotted against hirudin
concentration. Typically, the hirudin concentrations of the range
are biologically relevant hirudin concentrations. The compositions
provided herein can provide a linear correlation between clotting
time and a variety of hirudin concentrations, including 0 .mu.g/mL
to about 20 .mu.g/mL, 0 .mu.g/mL to about 15 .mu.g/mL, 0 .mu.g/mL
to about 12 .mu.g/mL, 0 .mu.g/mL to about 10 .mu.g/mL, 0 .mu.g/mL
to about 7.5 .mu.g/mL, or 0 .mu.g/mL to about 5 .mu.g/mL. The
compositions provided herein can provide a linear correlation
between clotting time and a variety of hirudin concentrations,
including 0 .mu.g/mL to 20 .mu.g/mL, 0 .mu.g/mL to 15 .mu.g/mL, 0
.mu.g/mL to 12 .mu.g/mL, 0 .mu.g/mL to 10 .mu.g/mL, 0 .mu.g/mL to
7.5 .mu.g/mL, or 0 .mu.g/mL to 5 .mu.g/mL.
[0090] Any of a variety of known methods for determining the
appropriate combination of components to achieve desired
coagulation properties can be used. The following represents an
exemplary procedure for arriving at an appropriate combination. One
skilled in the art will recognize that many variations of the
following procedure are possible, and are therefore contemplated
herein. Factor X compound-containing solutions can be serially
diluted. Factor V compound-containing solution can be combined with
the various dilutions of Factor X compound solution and Normal
Human Plasma (NHP). The Factor V compound solution also can be
combined with the various dilutions of Factor X compound solution
and Normal Human Plasma (NHP), where the NHP has added thereto an
anticoagulant such as heparin. The Factor X compound dilution with
the desired, or closest to desired, clotting properties can then be
further, iteratively refined, or can be selected as the basis for
combining the Factor X compound solution and the Factor V compound
solution.
[0091] A Factor X compound dilution with desirable coagulation
properties can be one that results in a clotting time of about
10-13 seconds or 10-13 seconds for normal plasma with no
anticoagulant added, and a clotting time of about 35-50 seconds or
35-50 seconds for normal plasma with 3 units/mL heparin added
thereto. If the Factor X compound dilution having a clotting time
of about 35-50 seconds or 35-50 seconds for normal plasma with 3
units/mL heparin, has a clotting time less than about 10 seconds
for normal plasma with no anticoagulant added, benzamidine-HCl can
be added to the Factor V compound solution until the no
anticoagulant added clotting time for that Factor X compound
dilution is about 10-13 seconds or 10-13 seconds.
[0092] A Factor X compound dilution with desirable coagulation
properties can be one that results in a clotting time of about
15-20 seconds or 15-20 seconds for normal plasma with no
anticoagulant added, and a clotting time of about 40-55 seconds or
40-55 seconds for normal plasma with 3 units/mL heparin added
thereto. If the Factor X compound dilution having a clotting time
of about 40-55 seconds or 40-55 seconds for normal plasma with 3
units/mL heparin, has a clotting time less than about 15 seconds or
15 seconds for normal plasma with no anticoagulant added,
benzamidine-HCl can be added to the Factor V compound solution
until the no anticoagulant added clotting time for that Factor X
compound dilution is about 15-20 seconds or 15-20 seconds.
[0093] Another example of a Factor X compound dilution with
desirable coagulation properties can be one that results in a
clotting time of about 13-17 seconds or 13-17 seconds for plasma
with no anticoagulant added, and a clotting time less than about
100 seconds or 100 seconds for plasma with 7.5 units/mL heparin. If
the Factor X compound dilution having a clotting time of less than
about 100 seconds or 100 seconds for plasma with 7.5 units/mL
heparin, has a clotting time less than about 13 seconds or 13
seconds for plasma with no anticoagulant added, Factor X compound
solution can be further diluted, or the amount of lipid or
detergent added to the Factor V compound solution can be reduced
until a Factor X compound dilution is achieved with a no
anticoagulant added clotting time about 13-17 seconds or 13-17
seconds and a clotting time of less than about 100 seconds or 100
seconds for plasma with 7.5 units/mL heparin.
[0094] Also contemplated herein, additional components can be added
in varying amounts in arriving at the desired coagulation
properties of the coagulation assay composition. Exemplary
additional components include multivalent cation and lipid or
detergent. For example, as increasing amounts of lipid or detergent
are added, decreasing amounts of Factor X compound can be added, as
is known in the art and exemplified in U.S. Pat. No. 4,851,336.
Thus, in one example, the Factor-V solution can contain varying
amounts of lipid or detergent, and these varying amounts can be
compared to different dilutions of Factor X compound solution to
arrive at a coagulation assay composition with the desired
properties. One skilled in the art, based on the teachings herein
and knowledge in the art will understand the relation between lipid
or detergent and Factor X compounds, and will be able to determine
suitable combinations of these components in achieving a desired
coagulation assay composition.
[0095] Additional compounds can be present in a composition with
one of the above compounds, or can, optionally be added in addition
to the above compounds. Selection of components to add, and amounts
of any added component, can be accomplished by the guidelines
provided herein and known in the art, according to the desired
coagulation properties of the resultant coagulation assay
composition. Exemplary additional compounds include multivalent
cation, lipid or detergent, reagent plasma, prothrombin complex,
fibrinogen, antithrombin III, Factor II, anticoagulant, and
combinations thereof.
[0096] Compositions also can be prepared that are directed toward
coagulation assays for a subject to whom has been administered
other anticoagulants, such as other anti-Factor X.sub.a or
anti-Factor II.sub.a compounds, for example, hirudin. Such
compounds can be prepared according to the guidelines provided
above with respect to heparin, where the heparin is substituted
with the other anticoagulant to be assayed. In one example, a
hirudin-directed coagulation assay composition can be developed
where the normal plasma or blood sample clotting time with no
hirudin added is about 13-17 seconds or 13-17 seconds and the
normal plasma or blood sample clotting time with about 7.5-10
.mu.g/mL or 7.5-10 .mu.g/mL added is less than about 100 seconds or
100 seconds. In another example, a hirudin-directed coagulation
assay composition can be developed where the normal plasma or blood
sample clotting time with no hirudin added is about 10-13 seconds
or 10-13 seconds and the normal plasma or blood sample clotting
time with about 3-5 .mu.g/mL added is about 35-50 seconds or 35-50
seconds. In another example, a hirudin-directed coagulation assay
composition can be developed where the normal plasma or blood
sample clotting time with no hirudin added is about 15-20 seconds
or 15-20 seconds and the normal plasma or blood sample clotting
time with about 3-5 .mu.g/mL added is about 40-55 seconds or 40-55
seconds.
[0097] 2. Exemplary Compositions
[0098] By way of example, and not by limitation, the following
exemplary compositions are provided. Variations according to the
guidance provided herein and the knowledge of the art will be
apparent to those skilled in the art. A diagram demonstrating
exemplary preparations for the two exemplary compositions below, is
provided in FIGS. 2, 3 and 4.
[0099] In one embodiment, a composition can contain components from
two solutions, the first solution containing Factor X.sub.a, and
the second solution containing Factor V. The Factor
X.sub.a-containing solution can also contain buffer components such
as Tris maleate, at pH 7.5, NaCl, BSA, PEG and lactose. In another
example, the Factor X.sub.a-containing solution can also contain
buffer components such as Tris maleate, at pH 7.5, NaCl, glycine,
BSA, PEG and lactose. The Factor X.sub.a-containing solution can
also contain a multivalent cation, such as calcium chloride. The
amount of each of these components in the Factor X.sub.a-containing
solution is provided herein elsewhere. The Factor V-containing
solution can contain a plasma fraction containing Factor V, where
this plasma fraction can optionally be characterized as being
substantially free of Factors II, VII, IX and X. The Factor
V-containing solution can also contain buffer components, such as
Tris maleate, at pH 7.5, NaCl, BSA, PEG and lactose. In another
example, the Factor V-containing solution can also contain buffer
components, such as Tris maleate, at pH 7.5, NaCl, BSA and PEG. The
Factor V-containing solution can also contain fibrinogen, such as
mammalian fibrinogen, including bovine fibrinogen. The Factor
V-containing solution can also contain lipid or detergent, such as
rabbit brain cephalin. The Factor V-containing solution can also
contain benzamidine HCl. The amount of each of these components in
the Factor V-containing solution is provided herein elsewhere.
[0100] In another embodiment, a composition can contain components
from two solutions, the first solution containing Factor X.sub.a,
and the second solution containing Factor V. The Factor
X.sub.a-containing solution can also contain buffer components such
as Tris maleate, at pH 7.5, NaCl, BSA, PEG and lactose. The Factor
X.sub.a-containing solution can also contain a multivalent cation,
such as calcium chloride. The Factor V-containing solution can
contain a plasma fraction containing Factor V, where this plasma
fraction can optionally be characterized as being substantially
free of Factors II, VII, IX and X. The Factor V-containing solution
can also contain buffer components, such as Tris maleate, at pH
7.5, NaCl, BSA, PEG and lactose. The Factor V-containing plasma
fraction can also contain plasma, such as mammalian plasma,
including normal human plasma or heparinized normal human plasma.
The Factor V-containing plasma fraction can also contain
prothrombin complex. The Factor V-containing plasma fraction can
also contain an anticoagulant, such as heparin. The Factor
V-containing solution can also contain lipid or detergent, such as
rabbit brain cephalin. The Factor V-containing solution can also
contain antithrombin III.
IV. Preparation of the Compositions
A. General Procedure Guidelines
[0101] The coagulation assay composition can be prepared by a
process that includes adding two or more components of the
composition under conditions in which the components do not react,
and after adding, maintaining the composition under one or more
different conditions under which the components do not react, until
the time of use. In one embodiment, not all components of the
composition can be present in an aqueous solution simultaneously
without some reaction occurring. Accordingly, methods provided
herein accomplish the addition of two or more components of the
composition under conditions in which the components do not react
by adding the components together under conditions in which not all
components are substantially present in aqueous solution
simultaneously. After such adding step, the composition is
maintained under conditions in which not all components are
substantially present in aqueous solution simultaneously, until the
time of conducting the coagulation assay. Typically the methods
provided herein include a method in which a Factor X
compound-containing solution and a Factor V compound-containing
solution are not both substantially present in aqueous solution
simultaneously.
[0102] In another embodiment, coagulation assay composition can be
prepared by a process that includes adding two or more components
of the composition under conditions in which the components are
substantially not admixed. For example, two aqueous solutions can
be added to a vessel, where the first solution can be placed in a
location or in a phase (e.g., solid phase) such that the first
solution is not admixed with the second solution when the second
solution is added to the vessel. In another example, two solids can
be added to a vessel, and the conditions can be maintained such
that the solids do not admix; such conditions include conditions
that prevent the solids from substantially dissolving into a liquid
or converting into liquid phase by, e.g., maintaining a cold
temperature or an environment free of liquid or gaseous solvent
material. In one such embodiment, a first and second component of
the composition are combined such that the first and second
compositions are not in fluid mixture with one another. In another
such embodiment, a first and second component of the composition
are combined such that the first and second compositions are not in
a fluid composition with one another. Typically the methods
provided herein include a method in which a Factor X
compound-containing solution and a Factor V compound-containing
solution are not both substantially present in aqueous solution
simultaneously.
[0103] The multiple components so combined can be maintained in a
form in which they do not react, are not substantially
simultaneously present in liquid aqueous solution, are not
substantially admixed, or are not substantially in fluid admixture,
until performance of a coagulation assay. Maintaining the multiple
components in such a form can be accomplished by maintaining
constant or similar conditions to the conditions under which the
components were combined, or can be accomplished by modifying the
conditions to different conditions under which the components do
not react, are not substantially simultaneously present in liquid
aqueous solution, are not substantially admixed, or are not
substantially in fluid admixture, until performance of a
coagulation assay. Exemplary modification of conditions includes
conditions in which any H.sub.2O present in the combined components
is removed from the components without substantially transitioning
to liquid form; such as by sublimation via, e.g.,
lyophilization.
[0104] In one method for accomplishing the adding steps in forming
the coagulation assay compositions provided herein, two or more
aqueous components are combined. For example, an aqueous Factor X
compound-containing solution, which can also contain one or more
additional components such as a divalent cation, can be combined
with an aqueous Factor V compound-containing solution, which can
contain one or more additional components such as fibrinogen,
prothrombin complex, anticoagulant, lipid or detergent, or plasma.
In such a method, the two components (the Factor X
compound-containing solution and the Factor V compound-containing
solution) are not both in aqueous liquid form when combined. For
example, a first aqueous component can be present in a vessel in
frozen, form. When the second component is added to the first
component, the second component does not substantially cause the
first component to transition to aqueous liquid form. This can be
accomplished by bringing the frozen first component to a
temperature of about -10.degree. C. or less, about -20.degree. C.
or less, about -40.degree. C. or less, about -60.degree. C. or
less, about -80.degree. C. or less, or about -100.degree. C. or
less, or -10.degree. C. or less, -20.degree. C. or less,
-40.degree. C. or less, -60.degree. C. or less, -80.degree. C. or
less, or -100.degree. C. or less. The second component can be added
to the frozen first component under conditions in which the second
component freezes at a rate fast enough to not substantially cause
the first component to transition to aqueous liquid form.
Temperatures at which the second component freezes at a rate fast
enough to not substantially cause the first component to transition
to aqueous liquid form include about -10.degree. C. or less, about
-20.degree. C. or less, about -40.degree. C. or less, about
-60.degree. C. or less, about -80.degree. C. or less, or about
-100.degree. C. or less, or -10.degree. C. or less, -20.degree. C.
or less, -40.degree. C. or less, -60.degree. C. or less,
-80.degree. C. or less, or -100.degree. C. or less. Typically, when
three or more components are added, the additional component(s) are
also added and frozen according to the same guidelines as the
freezing of the second component.
[0105] In some embodiments, all of the two or more components are
frozen on a short time scale. While not intending to be limited by
the following, shorter time frames for freezing are considered to
prevent aqueous solutions from transitioning to liquid form, and
also to be conducive to maintaining biomolecules in active, native
form. Accordingly, in some embodiments, freezing each of two or
more components of the coagulation assay composition on a short
time scale can produce a composition in which all components, while
in frozen form, retain most, substantially all, or all activity
relative to pre-frozen form, without having reacted with each
other. Typically, freezing on short time scales can be accomplished
by contacting small volumes of liquid with a low temperature, as is
known in the art. Exemplary volumes that can be used include about
1 mL or less or 1 mL or less, typically about 100 .mu.L or 100
.mu.L. Exemplary temperatures that can be used include -10.degree.
C. or less, about -20.degree. C. or less, about -40.degree. C. or
less, about -60.degree. C. or less, about -80.degree. C. or less,
or about -100.degree. C. or less, or -10.degree. C. or less,
-20.degree. C. or less, -40.degree. C. or less, -60.degree. C. or
less, -80.degree. C. or less, or -100.degree. C. or less. Any of a
variety of quick freezing methods known in the art can be used to
quickly freeze the component-containing liquids provided
herein.
[0106] In one example of the above description, 100 .mu.L of a
first solution containing Factor V compound and cephalin is
contacted with a glass vessel, where the contacting surface of the
glass vessel is about -90.degree. C. or -90.degree. C. Before
adding the second solution, the frozen first solution is maintained
in contact with the glass at -90.degree. C. until the frozen first
solution reaches about -90.degree. C. or -90.degree. C. Next, 100
.mu.L of a second solution, which contains Factor X.sub.a and
calcium chloride, is contacted with the glass vessel containing the
frozen first solution, and at a temperature of about -90.degree. C.
or -90.degree. C.
[0107] An exemplary apparatus for low temperature combination of
components of the composition includes a cooling block, for
example, an aluminum block cooled to the desired temperature. Such
a cooling block can be placed into the desired temperature for
sufficient time to adopt that temperature (e.g., overnight in a
freezer or on dry ice) or can have circulated within the block a
fluid that is at or below the desired temperature (e.g., liquid
nitrogen, gaseous nitrogen, ethanol from an ethanol/dry ice bath).
Any of a variety of methods known in the art for attaining and
maintaining a desired temperature can be used for combination of
components of the composition according to the teachings provided
herein.
[0108] When the first and second components are combined in a
vessel, the vessel can be any suitable vessel for containing the
components. In one embodiment, the vessel also is suitable for
subsequently performing coagulation assay methods. In another
embodiment, the vessel can contain a single compartment. In another
embodiment, the vessel can contain multiple compartments. In some
embodiments, the vessel can be a sterile vessel.
[0109] Further to the above example, after both solutions are
frozen and sufficiently cooled, the vessel is transferred to a
lyophilizer without permitting the solutions to return to liquid
state. Lyophilization can be performed at normal lyophilization
conditions until substantially all H.sub.2O is removed from the
vessel, for example, for about 8 hours or 8 hours. The vessel can
then be sealed under vacuum with a vapor-impermeable seal, and
maintained at room temperature, or cooler temperatures.
[0110] In some instances, the vessels containing the lyophilized
compositions will contain discrete masses, where substantially all
of the components of each discrete mass are components from only
one of the liquids that were flash-frozen. For example, a vessel
containing lyophilized composition can contain a first discrete
mass containing substantially only components from the Factor V
compound-containing solution, and a second discrete mass containing
substantially only components from the Factor X compound-containing
solution.
V. Measuring Anticoagulants
[0111] Also provided herein are methods of determining the amount
of anticoagulant in a sample. Typically the amount compound(s)
having anti-Factor X.sub.a and/or anti Factor II.sub.a activity can
be determined using the methods provided herein. In one embodiment,
the coagulation assay composition provided herein can be used in a
one-step procedure for performing the assay. Any of a variety of
general procedures for assaying coagulation known in the art can be
used in the coagulation assay methods provided herein. The
following guidelines are intended to exemplify assay procedures;
other assay procedures known in the art can also be used in
performing the assay.
A. General Protocol Guidelines
[0112] The coagulation assay composition provided herein can be
used in methods that determine the propensity of a sample from a
subject to coagulate. For example, the composition can be used to
determine the influence of compounds that inhibit Factor X.sub.a or
Factor II.sub.a in a subject's blood or plasma. In one example, the
composition can be used to determine the influence of heparin on
coagulation of a subject's blood or plasma. In another example, the
composition can be used to determine the influence of hirudin on
coagulation of a subject's blood or plasma. Generally, the method
is performed by adding a sample, such as blood or plasma, to the
coagulation assay composition, mixing the sample and the
composition, and monitoring the time between adding the sample to
the composition and a reaction endpoint such as clot formation.
Each step, such as adding, mixing and monitoring, can be performed
by any of a variety of methods known in the art. For example, the
assay can be performed by any of a variety of methods known in the
art for determining the propensity of a sample from a subject to
coagulate. Typically, the time monitored begins upon adding the
sample to the coagulation assay composition, and ends at an
endpoint such as clot formation. The results of the assay can be
used to determine the clotting time of the sample, the clotting
time of the sample relative to the clotting time of a reference, or
the amount of anticoagulant in the sample, as determined by
comparison to a calibration or reference curve.
[0113] 1. Sample Treatment
[0114] A sample used in the coagulation assay methods provided
herein can be any biological fluid from a subject that contains one
or more compounds that influence blood coagulation. Generally, the
sample is in liquid form when added to the coagulation assay
composition. Exemplary samples include, but are not limited to
blood, plasma, serum, interstitial fluid, and lymph. Typical
samples are blood and plasma.
[0115] A sample collected from a subject can be tested immediately
after collection, or can be stored and later tested. In one
example, a blood sample can be collected by clean venipuncture in a
plastic tube containing 3.8% sodium citrate, and used immediately
after collection, or stored, or treated in one or more steps, such
as centrifugation where the plasma is collected an assayed.
Typically, when a sample is stored for more than about 2 hours, the
sample can be treated in any manner known in the art for storing
such a sample, according to the sample type and time period over
which the sample is to be stored. Storage conditions can include
temperatures such as 4.degree. C. or below 0.degree. C. For
example, plasma can be stored at about 4.degree. C. for about 24
hours, or longer, as is known in the art. Storage conditions can
also include addition of one or more compounds to the sample that
are known in the art for preserving samples such as blood or plasma
samples, for extended time periods. As an alternative to, or in
combination with addition of one or more compounds, a sample can
have removed therefrom, one or more compounds; for example, a blood
sample can have removed therefrom its cellular components, leaving
only the plasma portion of the blood. Appropriate storage and
treatment methods can be selected according to the type of sample
and the length of storage time, as is known in the art. In some
embodiments, a sample stored at room temperature or below, can be
warmed to a temperature ranging from about room temperature to
about 37.degree. C., or room temperature to 37.degree. C., before
performing the assay. A warmed sample can be incubated at the
selected temperature for about 1 to about 10 minutes or 1 to 10
minutes. Similarly, the vessel in which the assay is to be
performed (e.g., the vessel containing the coagulation assay
composition) can be warmed to the selected temperature.
[0116] A sample can also have added thereto one or more compounds
or compositions, prior to performing the coagulation assay. For
example, a sample can have added thereto coagulation suppressing
compounds or solutions, as known in the art, for example, a sodium
citrate solution, a sodium or potassium oxalate solution, or EDTA.
A typical composition added to a sample is a 3.8% sodium citrate
solution.
[0117] 2. Adding Sample and Coagulation Assay Composition
[0118] The coagulation assay can be initiated by adding the sample
to a vessel containing the coagulation assay composition. As an
alternative, the coagulation assay composition can be added to a
vessel containing the sample. Upon adding the sample and
coagulation assay composition, the sample and coagulation assay
composition disperse and become admixed together. Typically, the
sample is a liquid sample and the coagulation assay composition is
a solid and, upon adding the liquid sample and coagulation assay
composition, the components of the coagulation assay composition
disperse into the liquid sample such that the components of the
sample can react with the components of the coagulation assay
composition. In assays that measure time periods, the addition of
the sample and coagulation assay composition typically represents
the starting point of the assay. Thus, upon adding the sample and
coagulation assay composition, a time measuring device, such as a
stopwatch or electronic timer, including a timer integral to a
device such as a coagulometer, can be started. Any of a variety of
methods for monitoring coagulation assay time periods are known in
the art, and can be used in the coagulation assay methods provided
herein.
[0119] Upon adding the sample and coagulation assay composition, if
desired, the sample and coagulation assay composition can be mixed
by any of a variety of methods known in the art. For example, the
sample and coagulation assay composition can be mixed by sealing
the vessel and inverting the vessel once or a plurality of times,
or the sample and coagulation assay composition can be mixed using
a vortex mixer. The mixing can be performed until most or
substantially all components are dispersed and/or dissolved. For
example, the sample can coagulation assay composition can be mixed
for a time period ranging from about 3 seconds to about 5 seconds
or 3 seconds to 5 seconds. Typically, the sample can coagulation
assay composition can be mixed for about 5 seconds or 5
seconds.
[0120] The amount of sample to be added to the coagulation assay
composition can be any of a wide range of volumes, according to the
assay design (e.g., amount of coagulation assay composition to be
used, device used in monitoring coagulation, convenience of sample
collection and treatment, desired accuracy of the assay, desired
time duration of the assay), as is known in the art. For example,
an assay can be designed to accommodate a sample volume of about 5
mL to about 0.1 mL, typically about 0.5 mL. In another example, an
assay can be designed to accommodate a sample volume of 5 mL to 0.1
mL, typically 0.5 mL.
[0121] 3. Monitoring the Assay
[0122] After adding and/or mixing the sample and coagulation assay
composition, the assay mixture can be monitored for an endpoint,
such as clot formation. Typically, the endpoint monitoring is
performed while observing the amount of time after the start of the
assay for the endpoint to be attained. In such instances, upon
arriving at the endpoint, the time period between the point of
adding the sample and coagulation assay composition to the endpoint
can represent the clotting time for the coagulation assay.
[0123] The endpoint to be monitored can be defined according to the
assay design, as is known in the art. Typically, the endpoint will
be marked by the formation of a solid clot. Any of a variety of
methods for monitoring an endpoint of a coagulation assay, such as
solid clot formation, are known in the art, including, but not
limited to, visual monitoring, fibrometer monitoring (BBL
Fibrosystems, Franklin Lakes, N.J.), electrochemical monitoring
(using e.g., an i-STAT analyzer, i-STAT Corp., East Windsor, N.J.),
magnetic particle mobility monitoring (using e.g., a Behnk
Thrombostat Coagulometer, Behnk Elektronic GmbH & Co,
Norderstedt, Germany), magnetic particle stationary position
monitoring (using, e.g., a KC1A Coagulometer, Amelung GMBH, Lemgo,
Germany), viscoelasticity monitoring (Sonoclot Analyzer, Sienco,
Inc., Morrison, Colo.); electromechanical meter (Hemo Tec or Hepcon
HMS Plus or ACT Plus, Medtronic Inc., Minneapolis, Minn.)). For
example, visual monitoring can be performed by tilting or inverting
the vessel containing the sample and coagulation assay composition
in order to visually observe the formation of a clot, as known in
the art. In another example, the assay can be conducted in a vessel
containing a magnetically responsive particle, such as a steel
ball, the movement of which can be driven and monitored by a device
having a magnetic field; when the motion of the particle falls
below a defined amount, this can indicate that the endpoint has
been reached.
[0124] 4. Results
[0125] The results typically are determined according to the time
period between the point of adding the sample and coagulation assay
composition, and the endpoint. Results can be reported in terms of
the time for the assay (e.g., as the amount of time between adding
the sample and the coagulation assay composition and clot
formation), or in relative terms of a ratio between sample assay
time and the assay time for a reference such as normal blood or
normal plasma. Results can also be reported in terms of units of
anticoagulant per unit volume, such as units/mL of Factor X.sub.a
inhibitor or units/mL of Factor II.sub.a inhibitor. For example,
results can be reported as units/mL of heparin or .mu.g/mL of
hirudin; this can be accomplished by comparing the assay time to a
reference curve of anticoagulant factor concentration plotted vs.
assay time.
[0126] In one embodiment, the clotting time and anticoagulant
concentration to be measured are linearly correlated. For example,
the methods provided herein can result in clotting times for normal
plasma or blood, which have added thereto any of a range of
anticoagulant concentrations, that form a straight line when
clotting time is plotted against anticoagulant concentration. The
methods provided herein can yield a linear correlation between
clotting time and anticoagulant for a variety of anticoagulants and
for a variety of anticoagulant concentrations. For example, when
the anticoagulant is heparin, the methods provided herein can
result in a linear relationship between clotting time and heparin
concentration for ranges such as 0 units/mL to about 20 units/mL, 0
units/mL to about 15 units/mL, 0 units/mL to about 12 units/mL, 0
units/mL to about 10 units/mL, 0 units/mL to about 7.5 units/mL, or
0 units/mL to about 5 units/mL. In another example, when the
anticoagulant is hirudin, the methods provided herein can result in
a linear relationship between clotting time and hirudin
concentration for ranges such as 0 .mu.g/mL to about 20 .mu.g/mL, 0
.mu.g/mL to about 15 .mu.g/mL, 0 .mu.g/mL to about 12 .mu.g/mL, 0
.mu.g/mL to about 10 .mu.g/mL, 0 .mu.g/mL to about 7.5 .mu.g/mL, or
0 .mu.g/mL to about 5 .mu.g/mL. In another example, when the
anticoagulant is heparin, the methods provided herein can result in
a linear relationship between clotting time and heparin
concentration for ranges such as 0 units/mL to 20 units/mL, 0
units/mL to 15 units/mL, 0 units/mL to 12 units/mL, 0 units/mL to
10 units/mL, 0 units/mL to 7.5 units/mL, or 0 units/mL to 5
units/mL. In another example, when the anticoagulant is hirudin,
the methods provided herein can result in a linear relationship
between clotting time and hirudin concentration for ranges such as
0 .mu.g/mL to 20 .mu.g/mL, 0 .mu.g/mL to 15 .mu.g/mL, 0 .mu.g/mL to
12 .mu.g/mL, 0 .mu.g/mL to 10 .mu.g/mL, 0 .mu.g/mL to 7.5 .mu.g/mL,
or 0 .mu.g/mL to 5 .mu.g/mL. When said linear relationship is
determined, the number of different heparin or hirudin
concentrations can be 2 or more, 3 or more, 4 or more, 5 or more or
6 or more, where typically most or all of the different heparin or
hirudin concentrations differ in concentration by the same
incremental amount (e.g., 0 units/mL, 5 units/mL, 10 units/mL, 15
units/mL and 20 units/mL).
B. Exemplary Protocol
[0127] By way of example, and not by limitation, the following
assay procedures are provided. Variations according to the guidance
provided herein and the knowledge in the art will be apparent to
those skilled in the art.
[0128] In a first example, fresh blood from a subject can be mixed
with a 3.8% sodium citrate solution in volume ratios 9:1
(blood:sodium citrate solution), to a total volume of 2 mL. A 0.5
mL aliquot of the blood sample is then added to a siliconized glass
vessel containing the coagulation assay composition, and the
solution is inverted by hand for 3-5 seconds, or until fully mixed.
Time monitoring of the reaction begins upon addition of the sample
to the composition. After mixing, the vessel is repeatedly inverted
and visually inspected for clot formation. Clot formation is
determined by formation of a solid clot in the vessel. Time
monitoring of the reaction ends upon clot formation.
[0129] In a second example, fresh blood from a subject can be mixed
with a 3.8% sodium citrate solution in volume ratios 9:1
(blood:sodium citrate solution), to a total volume of 2 mL. A 0.5
mL aliquot of the blood sample is then added to a siliconized glass
vessel containing the coagulation assay composition and a 2 mm
steel ball, and the solution is mixed for 3-5 seconds using a
vortex mixer. Time monitoring of the reaction begins upon addition
of the sample to the composition. Immediately after mixing, the
vessel is placed onto a Behnk Thrombostat coagulometer configured
to monitor the motion of the 2 mm steel ball in determining the
coagulation assay endpoint. The coagulometer monitors the motion of
the steel ball, and monitors the time elapsed until an endpoint,
such as clot formation, is reached. Clot formation is detected by
impairment of movement of the steel ball. Time monitoring of the
reaction ends upon clot formation.
[0130] The results of the assay can be reported as the amount of
time between introduction of the sample to the composition and clot
formation for the sample, or as a ratio of the amount of time
between introduction of the sample to the composition and clot
formation for the sample relative to the amount of time between
introduction of the sample to the reagent and clot formation for a
standard or reference, or can be units of anticoagulant/mL as
determined by comparing the clotting time to a reference curve.
C. Enhanced Clotting Inhibition
[0131] A subject sample can have added thereto an anticoagulant
such as heparin or hirudin, and then be subjected to the clotting
assay, as provided herein. Typically, several samples from a
subject are used or several aliquots of a sample from a subject are
used, where different samples can contain different amounts of the
anticoagulant, to thereby construct a curve of the clotting
response as influenced by added anticoagulant. In this assay, a
sample from a subject is mixed with a known amount of anticoagulant
such as heparin or hirudin, and also can be mixed with a
coagulation inhibiting solution such as sodium citrate. The
anticoagulant/sample mixture can then be added to the coagulation
assay composition, and the clotting assay performed according to
the general parameters provided herein.
[0132] The anticoagulant that can be used in performing such test
can be any anticoagulant known in the art to inhibit Factor X.sub.a
or Factor II.sub.a. Exemplary anticoagulants that can be used
include heparin and hirudin. Results from the test can be plotted
on a curve to determine the response of the subject to the
anticoagulant. As described herein, the curve can be a straight
line over a range of anticoagulant concentrations.
D. Standard Curve
[0133] Similar to the method of enhancing clotting inhibition of a
subject sample, a standard sample, such as normal human plasma can
have added thereto an anticoagulant such as heparin or hirudin, and
then be subjected to the clotting assay, as provided herein.
Typically, several standard samples or several aliquots of a
standard sample are used, where different samples can contain
different amounts of the anticoagulant, to thereby construct a
standard curve of the clotting response as influenced by added
anticoagulant. In this assay, a standard sample is mixed with a
known amount of anticoagulant such as heparin or hirudin, and also
can be mixed with a coagulation inhibiting solution such as sodium
citrate. The anticoagulant/sample mixture can then be added to the
coagulation assay composition, and the clotting assay performed
according to the general parameters provided herein.
[0134] As described herein, the curve can be a straight line over a
range of anticoagulant concentrations. Typically, when the curve is
a straight line, the range of anticoagulant concentrations includes
concentrations clinically relevant for the application. For
example, the range can be one relevant for screening subjects for
their blood's propensity to clot; one relevant for pre-operative
determination of the propensity of a subject's blood to clot; one
relevant for determination of the propensity of a subject's blood
to clot during an operation; one relevant for post-operative
determination of the propensity of a subject's blood to clot.
Exemplary operations for such use are open-heart and/or coronary
bypass operations. The range of concentration for which the
anticoagulant concentration and clotting time are linear can be
determined in the preparation of the coagulation assay composition,
as provided herein.
E. Properties of Methods
[0135] In one embodiment, the coagulation assay methods provided
herein can be performed to accurately determine anticoagulant
concentrations for a subject whose blood has undergone
hemodilution. Subjects undergoing surgery such as coronary bypass
surgery can have their blood diluted by a large percent, such as
about 25-60% or 25-60%. To such subjects, anticoagulants such as
heparin are routinely administered. However, dilution of the blood
can result in uncertainties as to the amount of anticoagulant
present in the subject's diluted blood when tested using methods
other than those provided herein (e.g., ACT test). The methods
provided herein can be used to accurately determine the
concentration of such anticoagulants. Methods are performed
substantially as provided above, and the clotting time can be used
to determine the concentration of anticoagulant in the sample. By
virtue of the linear correlation between clotting time and
anticoagulant concentration at clinically relevant concentrations
(e.g., heparin concentrations ranging from 0 units/mL to about 7.5
units/mL), the concentration of anticoagulant can be accurately
measured even after having undergone hemodilution and
administration of exogenous anticoagulant. Thus, the present
coagulation assay methods can be performed for a subject that has
undergone hemodilution, to determine the activity and/or
concentration of anti-Factor X.sub.a and/or anti Factor II.sub.a
compounds such as heparin or hirudin without the accuracy or
precision of the assay being influenced by hemodilution.
[0136] In another embodiment, the coagulation assay methods can be
performed to accurately determine the activity and/or concentration
of anti-Factor X.sub.a and/or anti Factor II.sub.a compounds
regardless of the presence of compounds such as aprotinin or
abciximab in a sample. Aprotinin can be administered to a subject
to reduce the incidence of post-operative abnormal bleeding and
blood transfusion due to activation of the fibrinolytic pathway.
However, aprotinin in a subject can interfere with the results of
some coagulation assay methods of samples from the subject. The
present coagulation assay methods can be performed to determine the
activity and/or concentration of anti-Factor X.sub.a and/or anti
Factor II.sub.a compounds such as heparin or hirudin, without
having the accuracy or precision influenced by the presence of
aprotinin in the sample.
[0137] In another embodiment, the coagulation methods can be
performed to accurately determine the activity and/or concentration
of anti-Factor X.sub.a and/or anti-Factor II.sub.a compounds
regardless of the temperature of the sample. In some instances, a
subject can be tested for clotting activity of the subject's blood
while the blood is kept at temperatures far below normal body
temperature (e.g., temperatures ranging from about 27.degree. C. to
about 22.degree. C. or 27.degree. C. to 22.degree. C.). Lower
temperatures of the sample can slow the assay time for some assay
methods. However, the present assay methods are not substantially
affected by lower temperatures of the sample. Further, standard
curves of normal plasma or blood can be established at any of a
variety of temperatures such that quantitative results can be
obtained notwithstanding the lower temperatures. Thus, the present
coagulation assay methods can be performed to determine the
activity and/or concentration of anti-Factor X.sub.a and/or anti
Factor II.sub.a compounds such as heparin or hirudin, without
having the accuracy or precision influenced by lower sample
temperatures.
[0138] In another embodiment, the coagulation methods can be
performed to accurately determine the activity and/or concentration
of anti-Factor X.sub.a and/or anti-Factor II.sub.a compounds in a
time frame and with sufficient simplicity so as to be used at the
point of care. In one example, the coagulation methods provided
herein can be performed while a subject is physically present at
clinic or other care-giving or diagnostic facility. In another
example, the coagulation methods provided herein can be performed
during the course of a surgical procedure, such as during open
heart surgery. In another example, the coagulation methods provided
herein can be performed immediately prior to surgery. In another
example, the coagulation methods provided herein can be performed
immediately subsequent to surgery. Such methods further can be
performed on a multiplicity of occasions, as the occasion requires.
For example, the blood of a subject undergoing a surgical procedure
can be tested a multiplicity of occasions, such as, but not limited
to, on two or more occasions during the course of the surgery, one
assay every designated time period (e.g., once every 30 minutes or
every hour), prior to surgery and during surgery, during surgery
and subsequent to surgery, on multiple occasions prior to surgery,
on multiple occasions subsequent to surgery, and combinations
thereof. In one embodiment, the composition formulated for point of
care coagulation assay methods is a composition in which the Factor
V compound is present in a plasma fraction which has had added
thereto prothrombin, plasma, fibrinogen and lipid or detergent, and
the Factor X compound can have added thereto a multivalent cation
salt. An example of a composition that can be used for point of
care applications is provided in FIGS. 2A, 2B, 3 and 4. In FIG. 2A,
Composition I can be combined with a Factor X compound-containing
composition to form a Point of Care Assay Composition. In FIG. 3, a
flow chart of an exemplary process for preparing Factor V
compound-containing composition and Factor X compound-containing
composition for preparation of a Point-of-Care coagulation assay
composition are presented.
[0139] In another embodiment, the coagulation methods can be
performed to accurately determine the activity in the blood of a
subject of anti-Factor X.sub.a and/or anti-Factor II.sub.a
compounds at different anti-Factor X.sub.a and/or anti-Factor
II.sub.a compound concentrations. A sample from a subject can have
added thereto one or more different concentrations of anti-Factor
X.sub.a and/or anti-Factor II.sub.a compound (e.g., heparin or
hirudin), a coagulation method can be performed for each
concentration, and the influence of the anti-Factor X.sub.a and/or
anti-Factor II.sub.a compound on the clotting time of the sample
can be determined. In another embodiment of such methods, the
subject can have added thereto one or more different concentrations
of anti-Factor X.sub.a and/or anti-Factor II.sub.a compound (e.g.,
heparin or hirudin), a coagulation method can be performed for each
concentration, and the influence of the anti-Factor X.sub.a and/or
anti-Factor II.sub.a compound on the clotting time of the blood of
the subject can be determined. An exemplary composition that can be
formulated for a coagulation assay methods that determine clotting
time at different concentrations of anti-Factor X.sub.a and/or
anti-Factor II.sub.a compound is a composition in which the Factor
V compound is present in a plasma fraction which has had added
thereto fibrinogen and lipid or detergent, and the Factor X
compound can have added thereto a multivalent cation salt. An
example of a composition that can be used for point of care
applications is provided in FIGS. 2A and 2B, where Composition II
of FIG. 2B can be combined with a Factor X compound-containing
composition to form a Heparin Does Response Assay Composition. In
FIG. 4, a flow chart of an exemplary process for preparing Factor V
compound-containing composition and Factor X compound-containing
composition for preparation of a Dose Response coagulation assay
composition are presented. Such methods can be used, for example,
to indicate that a subject may have an antithrombin III deficiency,
an anti-heparin antibody, or other medication that might reduce the
ability of heparin and/or hirudin to inhibit coagulation, when
clotting times are shorter than expected. Such methods also can be
used, for example, or to indicate that a subject may have
anticoagulant drugs, when clotting times are longer than
expected.
F. Variations of the Method
[0140] The methods provided herein can be used by adding a sample,
such as a blood sample, to a vial containing a lyophilized
coagulation assay composition provided herein. Also contemplated
herein, the methods can include adding a sample to a first liquid
containing Factor X compound and adding the sample/Factor X
compound combination to a liquid or solid containing Factor V
compound. Also contemplated herein, the methods can include adding
a sample to a first liquid containing Factor V compound and adding
the sample/Factor V compound combination to a liquid or solid
containing Factor X compound. Also contemplated herein, the methods
can include adding a sample to a first solid containing Factor X
compound and adding the sample/Factor X compound combination to a
liquid or solid containing Factor V compound. Also contemplated
herein, the methods can include adding a sample to a first solid
containing Factor V compound and adding the sample/Factor V
compound combination to a liquid or solid containing Factor X
compound. In such methods, Factor V compound-containing composition
and Factor X compound-containing composition typically are not
mixed prior to contacting either the Factor V compound-containing
composition or the Factor X compound-containing composition with
sample. In such methods, Factor X compound can be present in excess
relative to the amount of sample. Also in such methods, Factor V
compound can be present in excess relative to the amount of sample.
Also in such methods, multivalent cation salt can be present in
excess relative to the amount of sample. In such methods, sample
and Factor X compound-containing composition are typically not in
contact with each other for more than about 2 seconds or 2 seconds,
prior to contacting the sample/Factor X compound-containing
solution with Factor V compound.
VI. Combinations and Kits
[0141] Also provided herein are combinations and kits, wherein the
combinations and kits contain the coagulation assay composition
provided herein, in combination with one or more additional
constituents. Any of a variety of additional constituents can be
provided in combination with the compositions provided herein;
additional constituents can, for example, assist in performing a
coagulation assay, assist in interpreting the results of a
coagulation assay, or assist in constructing a reference for
comparison with a coagulation assay.
[0142] In one embodiment, the coagulation assay composition is
provided with one or more constituents that assist in performing
the assay. For example, the composition can be provided with a
vessel containing the composition. The vessel can be a vessel in
which the composition is stored for time periods between
preparation of the combinations and use of the combination. The
vessel can be a vessel in which the coagulation assay is to be
performed. The vessel also can be for both storage and performing
the assay. Typically, a vessel in which the composition is stored
will be sealed with a vapor-impermeable seal, or otherwise
configured to prevent liquid or gaseous H.sub.2O from entering the
vessel in which the composition is contained. Storage vessels can
be constructed of any of a variety of substances known in the art
for vapor impermeable storage of compositions, including, but not
limited to glass. In some instances, when the vessel is the vessel
in which the assay is to be performed, the portion of the vessel in
contact with the composition and blood or blood sample, typically
the inner surface of the vessel, can have a surface in which
aqueous solutions in contact with the surface have a high surface
tension. An example of such a high surface tension surface is
siliconized glass. Another example of such a high surface tension
surface is various plastics known in the art. In some embodiments,
the high surface tension surface such as siliconized glass will be
substantially homogenous; that is, the surface tension of the
entire surface is substantially the same.
[0143] In another embodiment, the combination can include an object
for monitoring coagulation of the sample. The object can be an
object that, upon application of a magnetic field, can be moved by
the magnetic field. Thus, objects can contain a substance sensitive
to magnetic fields, such as iron based substances, including steel.
The object can be configured such that, when the object is present
in a solution that does not contain a clot, the object can move
within the solution with relative ease, but when the object is
present in a solution that does contain a clot, object movement is
impaired to a large enough degree as to be measurable by a device
that can monitor such movements. Any of a variety of shapes for the
particle can be used, including but not limited to, spheres, cubes,
or irregularly shaped particles, provided that the particle
possesses the intended motile properties. An exemplary shape is a
ball. In one example, a steel ball can be used that has a diameter
of about 2 mm or 2 mm. The object, when present in a combination,
can be provided within the same compartment as the coagulation
assay composition (e.g., within the same vessel as the composition)
or in a separate compartment, where the object can be added with
the composition at the time of performing the coagulation
assay.
[0144] In another embodiment, a combination can include a device
for monitoring a coagulation assay endpoint, such as clot
formation. Any of a variety of known devices for monitoring
coagulation assays can be provided herein, including, but not
limited to a fibrometer (BBL Fibrosystems, Franklin Lakes, N.J.),
an electrochemical analyzer (e.g., the i-STAT analyzer from i-STAT
Corp., East Windsor, N.J.), a magnetic particle mobility monitoring
meter (e.g., the Behnk Thrombostat Coagulometer from Behnk
Elektronic GmbH & Co, Norderstedt, Germany), magnetic particle
stationary position monitoring (using, e.g., a KC1A Coagulometer,
Amelung GMBH, Lemgo, Germany), viscoelasticity monitoring meter
(e.g., the Sonoclot Analyzer from Sienco, Inc., Morrison, Colo.);
electromechanical meter (Hemo Tec or Hepcon HMS Plus or ACT Plus,
Medtronic Hemo Tec, Medtronic Inc., Minneapolis, Minn.). In one
embodiment, a combination can include device for monitoring
movement of the coagulation monitoring object within the sample.
Such a device can be a device that can form a magnetic field and/or
can monitor magnetic field influences of the coagulation monitoring
object. Any of a variety of devices for monitoring movement of a
magnetically influenceable object can be used, as known in the art.
In one example, such a device can contain a flat horizontal surface
upon which the sample can be placed, and below which is located
device components that can form a magnetic field and/or can monitor
magnetic field influences of the coagulation monitoring object. An
exemplary device is the Behk Thrombostat coagulometer.
[0145] In another embodiment, a combination can include a blood
collector and/or a blood sample container. The blood collector can
be any of a variety of apparatuses known in the art for the
collection of blood. As an integral or separate constituent, a
combination can contain a blood sample container, which can be any
object able to contain a liquid such as blood, typically a sterile
object. A blood sample container can also contain a coagulation
inhibitor, as are known in the art. An exemplary blood sample
container can contain a 3.8% sodium citrate solution, where the
volume of the sodium citrate solution can be about 1/10 or 1/10 the
final volume after blood is added to the sodium citrate
solution.
[0146] A kit can contain any of the combinations provided herein,
and also optionally additional components such as instructions for
use of the assay composition, information for interpreting the
results that can include a reference curve or table (e.g., a curve
in which reference amounts of heparin are plotted against clotting
time), liquid dispensing apparatuses (e.g., pipettes), a device for
mixing a vessel containing blood or a blood sample and the
coagulation assay composition (e.g., a vortex mixer), a device for
monitoring time elapsed during the coagulation assay (e.g., a
stopwatch).
[0147] A kit also can contain additional chemical components
separately packed from the coagulation assay composition. One
additional chemical component can be, for example, an anticoagulant
such as heparin or hirudin, which can be used for calibrating or
standardizing multiple coagulation assay compositions, or can be
used for addition into the sample or assay solution in determining
the influence of exogenous anticoagulant on the subject's sample,
or can be administered to a subject in monitoring the response of
the subject to the anticoagulant, or can be administered to a
subject therapeutically, if appropriate, after determining the
results of the coagulation assay. Another additional chemical
component can be, for example, plasma (e.g., Normal Human Plasma),
which can have as its source one or more subjects, and can
optionally contain added thereto, an anticoagulant such as heparin
or hirudin.
[0148] The packaging material used in the kit can be one or more
physical structures used to house the contents of the kit, and can
be constructed by well known methods, typically to provide a
contaminant-free environment. The packaging material can have a
label that indicates the components of the kit. In addition, the
packaging material contains instructions indicating how the
materials within the kit are employed to perform a coagulation
assay or to interpret the results of a coagulation assay.
Instructions typically include a tangible expression describing the
steps of performing the assay and/or at least one assay method
parameter, such as the amount of sample to add with the coagulation
assay composition, methods of mixing, and methods of monitoring
coagulation. The kit can include one or more containers capable of
holding within fixed limits the coagulation assay composition, or
buffer solution, or other composition used in the coagulation assay
methods. For example, a kit can include a glass vial used to
contain the coagulation assay composition.
[0149] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
[0150] Nine liters of bovine blood was anticoagulated with one
liter of 3.8% sodium citrate solution and then centrifuged at
2500.times.g to remove blood cells. To one liter of the resulting
plasma was added 7.35 g of trisodium citrate, which was stirred to
dissolve the citrate, followed by the addition of 20.825 g of solid
barium chloride at room temperature over a period of about 90
minutes. The heavy precipitate which formed was removed by
centrifugation of the plasma mixture at 3000.times.g for about 20
minutes, leaving a clear plasma supernatant. 270 g of solid
ammonium sulfate was added to the clear plasma supernatant at room
temperature and allowed to set for 90 minutes and then centrifuged
at 4000.times.g for 15 minutes, and a precipitated protein fraction
obtained. The recovered precipitated protein fraction was dissolved
in 350 ml of distilled water and then dialysed against a 0.9 NaCl
solution or tap water until all ammonium ions had been removed. The
dialysed plasma fraction was then clarified by centrifugation at
3000.times.g for 15 minutes at room temperature and then buffered
to a pH of 7.5 by mixing nine volumes of the dialysed plasma
fraction with one volume of a solution containing 2.5 g of
polyethylene glycol, 9.0 g of NaCl, 98.88 g of tris maleate,
dissolved in 1 liter of distilled water.
Example 2
Heptest HDR
Preparation of Plasma Fraction Mixture
[0151] One volume of the clarified, buffered plasma fraction
resultant from Example 1 is combined with one volume of 20% lactose
buffer and 100 mM tris maleate at pH 7.5, resulting in a mixture
termed for convenience of this example, mixture A. To mixture A is
added bovine fibrinogen to achieve a final concentration of added
fibrinogen of 3 mg/mL, resulting in a mixture termed for
convenience of this example, mixture B. To mixture B, cephalin
(prepared according to the method provided in Bell et al., Nature,
174:880 (1954)) is added in ratios that can range from about 6:0.5
(mixture B:cephalin) to about 6:1 (mixture B:cephalin) resulting in
a mixture termed for convenience of this example, mixture C.
Preparation of the Factor X.sub.a Solution
[0152] The Factor X.sub.a is prepared according to the method of
Yin et al. (Biol. Chem. 243:112 (1968)). Four aliquots of the
Factor X.sub.a solution are prepared by diluting Factor X.sub.a
with a buffer at ratios of: 1:5/1:10/1:20/1:30 (Factor
X.sub.a:buffer) where the buffer contains 20% lactose, 250 mM
calcium chloride, 1% PEG 6000, 1% BSA, and 100 mM Tris maleate at
pH 7.5. In alternative experiments, four aliquots of the Factor
X.sub.a solution are prepared by diluting Factor X.sub.a with a
buffer at ratios of: 1:5/1:10/1:20/1:30 (Factor X.sub.a:buffer)
where the buffer contains 5% lactose, 50 mM calcium chloride, 150
mM NaCl, 1% glycine, 0.1% PEG 6000, 3% BSA, and 100 mM Tris maleate
at pH 7.5. The resultant four solutions are different dilutions of
a solution termed the Factor X.sub.a solution.
Control Plasma
[0153] Two normal human plasma (NHP) samples are prepared; a first
with no added heparin, and a second with 3 units of bovine or
porcine heparin added per ml NHP.
Coagulation Assay
[0154] The four different dilutions of Factor X.sub.a solution are
then tested with mixture C using the two Control Plasmas in
coagulation assays. In performing the coagulation assays, 0.1 mL
mixture C, 0.1 mL Factor X.sub.a solution, and 0.5 mL Control
Plasma are mixed, and the time until clot formation is
monitored.
[0155] The appropriate dilution of Factor X.sub.a solution aliquot
that can be used as the basis for subsequent steps of freezing and
lyophilization is the dilution that gives a clotting time of about
10-13 seconds for Control Plasma with no heparin, and a clotting
time of about 35-50 seconds for Control Plasma with 3 units
heparin/mL. When the dilution of the Factor X.sub.a solution with
the clotting profile closest to the above is the most or least
diluted of the aliquots, additional aliquots can be prepared, as
needed, to determine an optimized dilution with the above
coagulation time profile for the two Control Plasmas. When the
dilution of the Factor X.sub.a solution with a clotting time within
about 35-50 seconds for Control Plasma with 3 units heparin/mL, has
a clotting time less than about 10 seconds for Control Plasma with
0 units heparin/mL, benzamidine HCl can be incrementally added to
mixture C in ratios of about 60:1 (mixture C:200 mM benzamidine
HCl), until the clotting time for Control Plasma with 0 units
heparin/mL is about 10-13 seconds. Alternatively, when the dilution
of the Factor X.sub.a solution with a clotting time within about
40-55 seconds for Control Plasma with 3 units heparin/mL, has a
clotting time less than about 15 seconds for Control Plasma with 0
units heparin/mL, benzamidine HCl can be incrementally added to
mixture C in ratios of about 60:1 (mixture C:200 mM benzamidine
HCl), until the clotting time for Control Plasma with 0 units
heparin/mL is about 15-20 seconds.
[0156] Upon determining the optimized dilution of Factor X.sub.a
solution and the optimized amount of benzamidine HCl to add,
mixture C is combined with benzamidine HCl according to the
optimized ratio, and Factor X.sub.a is diluted with buffer
according to the optimized Factor X.sub.a solution dilution. The
resultant mixture C and Factor X.sub.a solutions can then be used
to prepare the coagulation assay composition.
Example 3
Heptest POC
Preparation of Plasma Fraction Mixture
[0157] The clarified, buffered plasma fraction resultant from
Example 1 is combined with cephalin, normal human plasma,
prothrombin complex, heparin and buffer containing 20% lactose and
100 mM tris maleate at pH 7.5 in the following ratio: 30 mL of
Example 1 plasma fraction: 40 mL cephalin: 50 mL heparinized normal
human plasma (NHP with 1 unit/mL heparin): 2 mL prothrombin
complex: 60 mL buffer: 50-300 units heparin. The amount of heparin
to be used can be selected according to the desired clotting time.
The components can be added in any order. The resultant mixture is
termed for convenience of this example, mixture D.
Preparation of the Factor X.sub.a Solution
[0158] Factor X.sub.a solution and dilutions thereof are prepared
as provided in Example 2.
Control Plasma
[0159] Two normal human plasma samples are prepared; a first with
no added heparin, and a second with 7.5 units of heparin added per
ml NHP.
Coagulation Assay
[0160] The different dilutions of Factor X.sub.a solution are then
tested with mixture D using the two Control Plasmas in coagulation
assays. In performing the coagulation assays, 0.1 mL mixture D, 0.1
mL Factor X.sub.a solution, and 0.5 mL Control Plasma are mixed,
and the time until clot formation is monitored.
[0161] The appropriate dilution of Factor X.sub.a solution aliquot
that can be used as the basis for subsequent steps of freezing and
lyophilization is the dilution that gives a clotting time of about
13-17 seconds for Control Plasma with no heparin, and a clotting
time of less than about 100 seconds for Control Plasma with 7.5
units heparin/mL. When the dilution of the Factor X.sub.a solution
with the clotting profile closest to the above is the most or least
diluted of the aliquots, additional aliquots can be prepared, as
needed, to determine an optimized dilution with the above
coagulation time profile for the two Control Plasmas. When the
dilution of the Factor X.sub.a solution with a clotting time less
than about 100 seconds for Control Plasma with 7.5 units
heparin/mL, has a clotting time less than about 13 seconds for
Control Plasma with 0 units heparin/mL, further dilutions of the
Factor X.sub.a solution can be used, or mixture D can be prepared
where the amount of cephalin is reduced relative to the remaining
components. Modifications to the Factor X.sub.a solution and/or
mixture D, and further clotting test can be performed iteratively
until the clotting time for Control Plasma with 0 units heparin/mL
is about 13-17 seconds and the clotting time for Control Plasma
with 7.5 units heparin/mL is less than about 100 seconds.
[0162] Upon determining the optimized dilution of Factor X.sub.a
solution, and the optimized amount of cephalin to add to mixture D,
mixture D is combined according to the optimized cephalin ratio,
and Factor X.sub.a is diluted with buffer according to the
optimized Factor X.sub.a solution dilution. The resultant mixture D
and Factor X.sub.a solutions can then be used to prepare the
coagulation assay composition.
Example 4
[0163] A glass vial with a 2.4 mm steel ball is cooled to about
-90.degree. C. Into the -90.degree. C. glass vial is placed 100
.mu.l of the plasma fraction mixture resultant from Example 2 or
Example 3, and the plasma fraction mixture freezes instantly. The
vial is maintained at -90.degree. C. Next, 100 .mu.l of the Factor
X.sub.a solution resultant from Example 2 or Example 3 is added to
the -90.degree. C. glass vial, and the Factor X.sub.a solution
freezes instantly. The vial is then placed into a lyophilizer at
standard settings for about 8 hours. The vial is then sealed under
vacuum, and then removed from the lyophilizer. The vial can be
stored at room temperature.
Example 5
[0164] The vial containing the coagulation assay composition of
Example 2 or Example 3, as lyophilized and having a steel ball
added in Example 4, can be used in coagulation assays. A fresh
blood sample is added to a vial containing 3.8% sodium citrate,
where the ratio of blood to sodium citrate is 9:1. A 0.5 mL aliquot
of the blood/sodium citrate solution is dispensed into the vial
containing the coagulation assay composition and steel ball, and a
timer is started. The vial is placed on a vortex mixture for about
3-5 seconds to mix the blood and lyophilized coagulation assay
composition. The vial is then placed in a Behnk Thrombostat
Coagulometer that is configured to monitor the motion of the 2 mm
steel ball. The steel ball is monitored until its motion is
impaired by a solid clot formed in the vial. At the time of solid
clot formation, the timer is stopped, and the time period from
adding the blood mixture to the vial until solid clot formation is
noted as the resulting clotting time of the assay.
[0165] Standard calibration curves can be prepared by adding known
amounts of anticoagulant such as heparin or hirudin to blood or
NHP, and performing the above coagulation assay to obtain a series
of clotting times as a function of heparin concentration, as shown
in FIG. 1.
* * * * *